Compounds having antiviral activity

ABSTRACT

In order to provide the anti-retrovirus active compound with low anti-coagulant action and low cytotoxicity, compounds comprising glycoside or the salt thereof wherein lipid is linked to position 2 of sialic acid having all hydroxyl groups at positions 4, 7, 8 and 9 completely sulfated, or KDN (2-keto-3-deoxy-D-glycero-2-nononic acid) having all hydroxyl groups at positions 4, 5, 7, 8 and 9 completely sulfated are provided.

This is a continuation of application Ser. No. 09/984,920 filed Oct. 31,2001 now U.S. Pat. No. 6,541,461 which in turn is a Divisional ofapplication Ser. No. 09/180,618 filed Jan. 19, 1999, now U.S. Pat. No.6,337,390, which in turn in the U.S. National Stage of InternationalApplication No. PCT/JP97/01654 filed May 16, 1997. The entire disclosureof the prior application is hereby incorporated by reference herein inits entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a novel compound with the anti-viral activityand a drug and anti-viral agent containing said compound as theeffective ingredient, more specifically relates to a medicine andanti-HIV active compound comprising the sulfated nonulonic acid.

2. Description of the Related Arts

Azidothymidine (AZT) and dideoxy inosine (DDI) have been used as theAIDS remedy. These agents are to inhibit the reverse transcriptase ofHIV, bringing about apothanasic effect to patients, while posingproblems that the chronic administration of AZT causes myelopathy andthat the DDI administration results in side reactions such as acutepancreatitis and peripheral neuropathy. Furthermore, the use of eitherdrug eventually results in the generation of virus resistant to thesedrugs.

Recently, the sulfated polysaccharide has been expected as a promisingAIDS remedy. It is well known that polysulfated compounds such asdextransulfate (I) (Japanese Patent Laid-Open Publication No.Sho63-45233), polyvinyl alcohol sulfate (II) (Antimicrob. AgentsChemother. 34, 134-138 (1990)), oligosaccharide sulfate (Japanese PatentLaid-Open Publication No. Hei2-304025) inhibit the proliferation of HIV.These compounds are produced simply by binding the sulfate group topolysaccharides, oligosaccharides or organic polymeric molecules.

In addition, the sulfated modified cyclodextrin (III) is cyclodextrin towhich lipid-soluble groups such as aryl-, alkyl-groups are introduced,and also has the proliferation inhibiting activity against retroviruses,HIV in particular (Japanese Patent Laid-Open Publication No.Hei4-136001).

In addition, as a virucide comprising the sulfated polysaccharide as theactive ingredient is disclosed the one using an acyl derivative of asulfated oligosaccharide glycoside (Japanese Patent Laid-OpenPublication No. Hei6-256373).

At first, it was conceived that the virucidic activity of sulfatedpolysaccharides was due to their reverse transcriptase inhibitingactivities, and now it is thought to be due to the interaction betweenthe sulfated polysaccharide and the coat protein gp120 of HIV.

However, since sulfated polysaccharides generally have the inhibitoryactivity for the blood coagulation system (anticoagulant activity), theyhave not been accepted as the suitable medicine.

DISCLOSURE OF THE INVENTION

This invention has been developed in view of the above-mentionedproblems, aiming at providing the anti-viral compound with a relativelyweak anti-blood coagulating activity and a low cytotoxicity, especiallythe one with the anti-HIV activity.

The present inventors have actively pursued the study to resolve theabove-mentioned problems, establishing that the following compounds areuseful for attaining the above-described purpose and completing thepresent invention.

(1) A compound having all hydroxyl groups of the sugar moiety ofglycoside comprising monosaccharide-lipid sulfated or the salt thereof,wherein said lipid moiety is bound to the anomeric position of saidsugar moiety.

(2) The compound or the salt thereof according to (1), wherein saidsugar and lipid moieties are connected by the O-glycosidic linkage orthe S-glycosidic linkage.

(3) The compound or the salt thereof according to (2), wherein saidlipid is linear, and said linear lipid has a branched structure.

(4) A compound or the salt thereof, wherein all hydroxyl groups ofnonuloic acid moiety of the glycoside comprising a monosaccharide-lipidwith said lipid bound to the anomeric position of nonuroic acidderivative are sulfated.

(5) A compound or the salt thereof, wherein all hydroxyl groups ofsialic acid moiety or KDN moiety of the glycoside comprising amonosaccaride-lipid with said lipid bound to the anomeric position ofsialic acid or KDN are sulfated.

(6) The compounds or the salts thereof according to (5), wherein thebinding of said sialic acid moiety or KDN moiety to the lipid moiety iseither the O-glycosidic of S-glycosidic linkage at position 2 of saidmoiety or the amidic linkage at position 1 of said residue.

(7) The compound or the salt thereof according to (6), wherein saidlipid is a linear lipid, and this lipid has a branched structure.

(8) The compound or the salt thereof according to (7), wherein saidbranched chain is localized at position 2 of the main chain of saidlipid moiety.

(9) The compound or the salt thereof according to (8), wherein saidlipid moiety has a forked two chain structure due to said branching.

(10) The compound or the salt thereof according to (9), wherein saidlipid moiety has an alkyl group with the skeleton-forming carbon atomsfrom 1 to 4 at said branching site.

(11) The compound or the salt thereof according to (9) or (10), whereinthe total number of said lipid skeleton-forming atoms is from 22 to 60.

(12) The compound or the salt thereof according to (11), wherein saidbranched chain comprises a carbon-carbon unsaturated bond.

(13) The compound or the salt thereof according to (11), wherein saidbranched chain is linear.

(14) The compound or the salt thereof according to (11), wherein saidbranched chain has an ester linkage or ether linkage, respectively.

(15) The compound or the salt thereof according to (14), wherein saidester linkage or ether linkage is localized at positions 1 or 2 of saidbranched chain.

(16) The compound or the salt thereof according to (11), wherein thenumber of skeleton-forming atoms is from 10 to 28 per one branchedchain.

(17) The compound or the salt thereof according to (16), wherein thenumber of skeleton-forming atoms is from 18 to 26 per one branchedchain.

(18) The compound or the salt thereof according to (17), wherein thenumber of skeleton-forming atoms is from 24 per one branched chain.

(19) The compound or the salt thereof according to (18), wherein saidforked branched chains are of the same length, respectively.

(20) The compound or the salt thereof according to (19), wherein saidforked branched chains are of the same, respectively.

(21) The compound or the salt thereof according to (20), wherein saidbranched chain has the ester bond or the ether linkage at its position 1or 2.

(22) The compound or the salt thereof according to (21), wherein saidbranched chain is linear.

(23) A medicine comprising the compound according to anyone of (1) to(3) in a pharmaceutically effective dosage.

(24) An antiviral drug containing the compound according to anyone of(1) to (3) in a pharmaceutically effective dose.

(25) An anti-HIV drug containing the compound according to anyone of (1)to (3) in a pharmaceutically effective dose.

(26) A drug containing the compound according to anyone of (1) to (21)in a pharmaceutically effective dose.

(27) An antiviral drug containing the compound according to anyone of(1) to (21) in a pharmaceutically effective dose.

(28) An anti-HIV drug containing the compound according to anyone of (1)to (21) in a pharmaceutically effective dose.

Drugs (23 and 26), furthermore, antiviral drugs (24 and 27), especiallyanti-HIV drugs (24 and 27) containing compounds according to any one ofthe above-described (1) to (22) are within the scope of the presentinvention. However, since drugs, anti-viral drugs and anti-HIV drugscontaining the compounds according to any one of the above-described (5)to (22) in pharmaceutically effective doses have lower anti-coagulantactivity as well as lower biological toxicity than those containingcompounds according to any one of the above-described (1) to (3), theformer group of drugs are pharmaceutically useful. It has been confirmedthat, of the above-described compounds, particularly, those having 24skeleton-forming atoms per one branched chain (24), furthermore, each ofsaid branched chains of which has the same structure, is linear and hasthe ether bond at its position 1 or 2, are pharmaceutically useful(examples 12, 13, 29, 50 and 59).

And, the usage of drugs, anti-viral drugs and anti-HIV drugs containingthe compound according to any one of the above-described (1) and (22) asthe drugs, anti-viral drugs and anti-HIV drugs, and the method fortreating the particular diseases (viral disease and acquired immunedeficiency syndrome caused by HIV) are also within the scope of thepresent invention.

BEST MODE FOR CARRYING OUT THE INVENTION

Definition of the Terms

As used in this specification, by the term “nonuloic acid” is meant thesame nonuloic acid as used generally, an acidic carbohydrate having acarboxyl group at its position 1 and 9 carbon atoms. Accordingly,“nonuloic acid derivatives” used in this specification includeneuraminic acid (5-amino-3,5-dideoxy-D-glycero-D-galacto-nonuloic acid)and neuraminic acid derivatives. Since the later-described “sialic acid”is an acyl derivative of neuraminic acid, it is also included in“nonuloic acid derivatives”.

By “KDN” used in this specification is meant2-keto-3-deoxy-D-glycero-2-nononic acid. And, by “sialic acid used inthis specification is meant the generic name for a series of derivativesof substances having the neuraminic acid as the basic structure (YasuoInoue, Seitai Bunsi no Kagaku, Tositu no Kagaku (Chemistry ofBiomolecules 1, Chemistry of Carbohydrates), p80-81, Baifukan), and acylderivatives, more specifically, such as N-acetylneuramic acid andN-glycolylneuramic acid are also included therein.

By “forked two chains” (for example, (9)) is meant a structureconstructed with two chains having more than 7 skeleton-forming atoms.Accordingly, alkyl groups according to (10), wherein the total number oftheir skeleton-forming atoms is 1 to 4, are not included in “chain”constructing “forked two chains”.

By “skeleton-forming atoms” is meant atoms constructing the skeleton ofthe chain, including carbon atom, oxygen atom, nitrogen atom, sulfuratom, etc. However, monovalent atom, such as hydrogen atom, is notincluded in the “skeleton-forming atoms”, because it cannot constructthe skeleton portion of the chain.

By “salt” is meant the sodium and potassium salts which are required toneutralize the intramolecular carboxylic acid and sulfonic acid, towhich cations bind so as not to decrease the biological activity. As tothe cations bound to said acids, any cations which do not lower thebiological activity of the compounds related to the present inventionmay be used.

Sugars

In principle, the compound related to this invention is a glycosidiccompound and the salt thereof comprising a sugar-lipid with said lipidbound to the anomeric carbon of said sugar, wherein hydroxyl groups ofsaid sugar moiety are all sulfated, having an excellent anti-retroviralactivity (the above-described (1)). Although there have been hithertofound the sulfated sugars with the anti-retroviral activity, theglycoside comprising a sugar-lipid with sulfate group introduced andhaving the anti-retroviral activity has not been discovered. In thisrespect, this invention is valuable in finding that a glycosidecomprising a sugar-lipid with the sulfate groups filly (100%) introducedor the derivative thereof has an excellent anti-viral activity.

When the sugar moiety of said glycoside is sialic acid or KDN, theglycoside not only has the strong anti-viral activity, but also lowercytotoxicity, and can preferably achieve the main purpose as medicine(the above-described (5) to (22), (26) to (28)). And, in this case, thehydroxyl groups of the sugar moiety of the compound related to thepresent invention to be all sulfated to form the wholly sulfatednonulonic acid (the above-described (4)) are at positions 4, 7, 8 and 9when nonulonic acid is N-acetylneuraniic acid, and the glycolyl hydroxylgroup at position 5, in addition to the hydroxyl groups at the positions4, 7, 8 and 9. In the case wherein nonulonic acid is KDN, all thehydroxyl groups at positions 4, 5, 7, 8 and 9 are sulfated.

The bond between the monosaccharide moiety and lipid moiety of thecompound related to the present invention can be of any type.Accordingly, the bond between the monosaccharide and lipid may be notonly the O-glycosidic linkage but also S-glycosidic linkage.Furthermore, in the case where the sugar moiety of the compound relatedto the present invention is nonuloic acid, the ainide linkage and esterlinkage can be formed using the carboxyl group at position 1, inaddition to a glycosidic linkage with the carbon atom at position 2. Thebond between monosaccharide and lipid in the compound related to thepresent invention can be such amide linkage and ester linkage.Therefore, although, by “glycoside” is generally meant a compoundwherein the monosaccharide and lipid moieties are linked by a glycosidiclinkage, by “glycoside” in this specification is meant a compoundwherein the monosaccharide and lipid are bonded not only in a glycosidiclinkage but also in amide and ester linkages. Accordingly, not onlycompounds having a glycosidic linkage at position 1 of monosaccharidesuch as glucose or position 2 of nonulonic acid but also those having anamide linkage or ester linkage at position 1 of nonulonic acid are alsoin the scope of “glycoside” of the present invention.

However, in consideration of the overall manufacturing easiness andbiological activity of these compounds, glycosides with theO-glycosidic, S-glycosidic, and amide linkages are preferred.

Lipids

By “lipid” in the compound related to this invention is meant the lipidin a broad sense including steroid, carotinoid, terpenoid, etc., andconceptually even the compound such as cholesterol. However, thecompound related to this invention is preferably a linear lipid, whichfurther preferably has the branched chain structure (the above-described(7)). The branch can be two-forked or three-forked, located at position2 of the main chain of the lipid moiety. As a result, the lipid ispreferably two-forked at the β position (β position with respect to thesugar moiety) of said lipid moiety (the above-described (8) and (9)).Furthermore, this branching site may have alkyl group with theskeleton-forming atoms 1 to 4 (the above-described (10)). “Alkyl groupwith the skeleton-forming atoms 1 to 4” herein cited includes, forexample, methyl group, ethyl group, propyl group etc.

The above-described two-forked chain can be a hydrocarbon chain, whichcan include heteroatoms such as oxygen, nitrogen, sulfur, etc.Furthermore, regardless of the species of component atoms, the totalnumber of the skeleton-forming atoms of lipid is preferably 22 to 60(the above-described (11)). In addition, the above-described two-forkedchains can have unsaturated bond between carbon atoms, respectively (theabove-described (12)). Also, although the above-described two-forkedchain can be further branched, they are preferably linear (theabove-described (13)).

In the case where the above-described two-forked chain containsheteroatoms as the component atom, each branched chain preferablycontains an ester bond or ether bond (the above-described (14)),furthermore, said ester bond or ether bond is preferably localized atposition 1 or 2 of said branched chain (the above-described (15)).

Herein, when the ester bond or ether bond is present at position 1 ofthe branched chain, the compound related to this invention will becomethe sulfated derivative of sialoglycerolipid with the excellentanti-retroviral activity (In this connection, in the case of ether bondpresent, said compound will be an alkyl glycerol wherein a long-chainalcohol is linked to the glycerol residue, and in the case of ester bondpresent, said compound will be amyl glycerol.). Furthermore, in the casewhere ester bond or ether bond is located at position 2 of the branchedchain, the glycerol residue in the glycerol area is suitably modified tobecome pseud-glycerol.

As to the length of branched chains, the number of skeleton-forming atomis preferably 10 to 28 (the above-described (18)), more preferably 18 to26 (the above-described (17)), most preferably 24 (the above-described(18)). In addition, forked-chains can be of different lengths, butpreferably of the same length (the above-described (19)), mostpreferably of the same structure comprised of the same component atoms(the above-described (20)).

Method of Administration

When compounds related to the present invention are used astherapeutics, they are administered singly or in combination withpharmaceutically acceptable medical carriers, either organic orinorganic and either solid or liquid. Their compositions are determinedaccording to the solubility, chemical property, administration route andschedule of compounds.

Compounds related to this invention can be administered by any suitabledesired administration routes. More specifically, compounds related tothis invention can be administered intraperitoneally, subcutaneously,percutaneously, intravenously or intra-arterially, and locally injectedin the case of animals, and intravenously, intra-arterially, by localinjection, intraperitoneally/intrapleurally, orally, subcutaneously,intramuscularly, sublingually, percutaneously, inhalationally orrectally in the case of humans.

Dosage Form

When compounds related to the present invention are administered as thedrug, they can be administered, according to the method and purpose oftheir administration, in the form of injection, suspension, tablet,granule, powder, capsule, ointment, cream, suppository, tape, etc. Forpreparing these drugs, solvent, solubilizing agent, isotonizing agent,preservative, anti-oxidant, excipient, binder, lubricant, stabilizer,etc. can be added.

Solvents are exemplified, for example, by water, physiological sodiumchloride solution, etc.; solubilizing agents, for example, by ethanol,polysorbates, chromophore, etc.; excipients, for example, by lacticacid, sucrose, starch, cellulose, crystalline cellulose, dextrin,mannitol, maltose, kaolin, calcium hydrogenphosphate, light anhydroussilicic acid, calcium carbonate, etc.; binders, for example, by starch,polyvinylpyrrolidone, hydroxypropyl cellulose, ethyl cellulose,carboxymethyl cellulose, gum arabic, etc.; disintegrators, for example,by starch, calcium carboxymethyl cellulose, etc.; lubricants, forexample, by magnesium stearate, talc, hydrogenated oil, etc.;stabilizers, for example, by lactose, mannitol, maltose, polysorbates,macrogols, polyoxyethylene hydrogenated castor oil, etc. Furthermore,glycerol, dimethylacetamide, 70% sodium lactate, surfactant, basicsubstances (such as sodium hydroxide, ethylenediamine, ethanolamine,sodium carbonate, arginine, meglumine, tris-aminomethane) may be added,if needed. Using these components, compounds related to this inventioncan be prepared to the dosage forms such as injections, tablets,granules, capsules, etc.

Compounds related to this invention can be administered orally in dosageforms such as granule, fine granule, powder, tablet, heavy syrup, softcapsule, syrup, emulsion, suspension, liposome, liquid preparation, etc.Liquid preparations such as emulsion, syrup, suspension, liquid drug,etc. for oral administration comprise generally used inert diluents, forexample, water or vegetable oil. These preparations can also includesupplements, for example, moistening agents, suspending agents,sweetening agents, aromatics, coloring matters, preservatives, etc., inaddition to these inert diluents. Liquid preparations may be envelopedin capsules made of absorbable materials such as gelatin.

For administration by intravenous, intramuscular and subcutaneousinjections, compounds may be made in powder form for injection, andprepared for injection prior to use. Solvents or suspending agents forpreparing drugs for parenteral administration, that is, injections, etc.are exemplified, for example, by water, propylene glycol, polyethyleneglycol, benzylalcohol, ethyl oleate, lecithin, etc. Preparation ofpharmaceutics can be carried out according to the conventional method.

Treatment

It is desirable that clinical dosages are appropriately increased and/ordecreased according to the age, symptom, presence or absence ofsimultaneous administration of other drugs. The daily dosage ofcompounds related to this invention may be administered once daily, orin two or three divided portions at appropriate intervals, orintermittently. In view of results of animal experiments and variousconditions, dosages of compounds related to this invention aredetermined such that the total dosage does not exceed a certain limitregardless of whether they are administered once or repeatedly. Needlessto say that the optimum dosage is varied according to the method ofadministration, conditions of patients or animals to be treated such asage, body weight, sex, susceptibility, food (diet), time ofadministration, drugs in joint use, patient's conditions, severity ofsymptoms, etc. In addition, the optimal dosage and administrationfrequency under the certain conditions must be determined by thesuitable usage determination test performed by a medical specialistaccording to the above-describe guideline.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphic representation of the dependency of biologicalactivity of two-forked alkylglycerol with a glycosidic linkage on thechain length

FIG. 2 is a diagram showing the reaction pathway from Examples 1 to 6.

FIG. 3 is a diagram showing the reaction pathway from Examples 7 to 12.

FIG. 4 is a diagram showing the reaction pathway from Examples 13 to 17.

FIG. 5 is a diagram showing the reaction pathway from Examples 18 to 21.

FIG. 6 is a diagram showing the reaction pathway from Examples 22 to 25.

FIG. 7 is a diagram showing the reaction pathway from Examples 26 to 29.

FIG. 8 is a diagram showing the reaction pathway from Examples 30 to 34.

FIG. 9 is a diagram showing the reaction pathway from Examples 35 to 36.

FIG. 10 is a diagram showing the reaction pathway from Examples 37 to41.

FIG. 11 is a diagram showing the reaction pathway from Examples 42 to45.

FIG. 12 is a diagram showing the reaction pathway from Examples 46 to51.

FIG. 13 is a diagram showing the reaction pathway from Examples 52 to56.

FIG. 14 is a diagram showing the reaction pathway from Examples 57 to60B.

FIG. 15 is a diagram showing the reaction pathway from Examples 60C to63.

FIG. 16 is a diagram showing the reaction pathway from Examples 64 to68.

EXAMPLES

Synthesis of Compounds

Example 1

Sodium[methyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosid]onate

Methyl5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosidonate[Chem. Ber., 99, 611 (1966)] (200 mg, 0.58 mmol) and sulfurtrioxide-trimethylamine complex (1600 mg, 11.5 mmol) were stirred inanhydrous dimethylformamide (5.8 ml) under the argon atmosphere at80-85° C. for 2 h. The reaction mixture was purified directly by silicagel column chromatography (gel 209 g, chloroform/methanol/water, 5:4:1),and further treated with Dowex 50W-X8 (Na form) resin. The product wasfurther purified by gel chromatography (Sephadex G-25, 400 ml, water) toobtain the title compound (359 mg, 82%) as white solid.

¹H-NMR (D₂O) δ: 3.37 (s, 3H, OCH₃), 2.88 (dd, 1H, J=4.0, 11.7 Hz,H-3eq), 1.97 (s, 3H, NAc), 1.77 (t, 1H, J=12.1 Hz, H-3ax).

Example 2

Sodium[methyl 5-acetamido-3,5-dideoxy4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-β-D-galacto-2-nonulopyranosid]onate

Methyl5-acetamido-3,5-dideoxy-D-glycero-β-D-galacto-2-nonulopyranosidonate[Chem. Ber., 99, 611 (1966)] (200 mg) was reacted by the generalprocedure according to Example 1 to obtain the title compound (218 mg,50%) as white solid.

¹H-NMR (D₂O) δ: 3.33 (s, 3H, OCH₃), 2.57 (dd, 1H, J=5.1, 13.2 Hz,H-3eq), 1.96 (s, 3H, NAc), 1.90 (t, 1H, J=13.2 Hz, H-3ax).

Example 3

3-O-[Sodium{5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosyl}onate]-1,2-di-O-hexyl-Sn-glycerol

3-O-[Sodium(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl)onate]-1,2-di-O-hexyl-Sn-glycerol[Japanese Patent Laid-Open Publication No. Hei1-125394] (19 mg) wasreacted by the general procedure according to Example 1 to obtain thetitle compound (15 mg, 46%) as white solid.

¹H-NMR (CD₃OD) δ: 3.02 (dd, 1H, J=4.9, 12.6 Hz, H-3eq), 1.92 (s, 3H,NAc), 1.83 (t, 1H, J=12.1 Hz, H-3ax), 1.39-1.26 (m, 12H, 6CH₂), 0.90 (t,6H, J=6.6 Hz, 2CH₂CH₃).

Example 4

3-O-[Sodium{5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-β-D-galacto-2-nonulopyranosyl}onate]-1,2-di-O-hexyl-Sn-glycerol

3-O-[Sodium(5-acetamido-3,5-dideoxy-D-glycero-β-D-galacto-2-nonulopyranosyl)onate]-1,2-di-O-hexyl-Sn-glycerol[Japanese Patent Laid-Open Publication No. Hei1-125394] (26 mg) wasreacted by the general procedure according to Example 1 to obtain thetitle compound (22 mg, 48%) as white solid.

¹H-NMR (CD₃OD) δ: 2.83 (dd, 1H, J=5.1, 12.8 Hz, H-3eq), 2.03 (s, 3H,NAc), 1.87 (t, 1H, J=12.3 Hz, H-3ax), 1.37-1.34 (m, 12H, 6CH₂), 0.90 (t,3H, J=6.8 Hz, CH₂CH₃), 0.90 (t, 3H, J=7.0 Hz, CH₂CH₃).

Example 5

3-O-[Sodium{5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosyl}onate]-1,2-di-O-decyl-Sn-glycerol

3-O-[Sodium(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl)onate]-1,2-di-O-decyl-Sn-glycerol[Japanese Patent Laid-Open Publication No. Hei1-125394] (190 mg) wasreacted by the general procedure according to Example 1 to obtain thetitle compound (268 mg, 88%) as white solid.

¹H-NMR (D₂O) δ: 2.94 (dd, 1H, J=5.0, 13.0 Hz, H-3eq), 1.99 (s, 3H, NAc),1.96 (t, 1H, J=12.5 Hz, H-3ax), 1.44-1.21 (m, 28H, 14CH₂), 0.89 (t, 6H,J=6.8 Hz, 2CH₂CH₃).

Example 6

3-O-[Sodium{5-acetamido-3,5-dideoxy4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-β-D-galacto-2-nonulopyranosyl}onate]-1,2-di-O-decyl-Sn-glycerol

3-O-[Sodium(5-acetamido-3,5-dideoxy-D-glycero-β-D-galacto-2-nonulopyranosyl)onate]-1,2-di-O-decyl-Sn-glycerol[Japanese Patent Laid-Open Publication No. Hei1-125394] (197 mg) wasreacted by the general procedure according to Example 1 to obtain thetitle compound (240 mg, 76%) as white solid.

¹H-NMR (D₂O) δ: 2.67 (dd, 1H, J=5.0, 13.0 Hz, H-3eq), 2.01 (s, 3H, NAc),1.93 (t, 1H, J=12.2 Hz, H-3ax), 1.42-1.20 (m, 28H, 14CH₂), 0.88 (t, 6H,J=6.8 Hz, 2CH₂CH₃).

Example 7

3-O-[Sodium{5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosyl}onate]-1,2-di-O-tetradecyl-Sn-glycerol

3-O-[Sodium(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl)onate]-1,2-di-O-tetradecyl-Sn-glycerol[Japanese Patent Laid-Open Publication No. Sho59-164798] (406 mg) wasreacted by the general procedure according to Example 1 to obtain thetitle compound (479 mg, 78%) as white solid.

¹H-NMR (CD₃OD) δ: 3.01 (dd, 1H, J=4.9, 11.9 Hz, H-3eq), 1.93 (s, 3H,NAc), 1.74 (t, 1H, J=12.1 Hz, H-3ax), 1.38-1.25 (m, 44H, 22CH₂), 0.90(t, 6H, J=6.8 Hz, 2CH₂CH₃).

Example 8

3-O-[Sodium{5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycerol-β-D-galacto-2-nonulopyranosyl}ornate]-1,2-di-O-tetradecyl-Sn-glycerol

3-O-[Sodium(5-acetamido-3,5-dideoxy-D-glycero-βα-D-galacto-2-nonulopyranosyl)ornate]-1,2-di-O-tetradecyl-Sn-glycerol[Japanese Patent Laid-Open Publication No. Sho59-164798] (87 mg) wasreacted by the general procedure according to Example 1 to obtain thetitle compound (120 mg, 91%) as white solid.

¹H-MR (CD₃OD) δ: 2.83 (DD, 1H, J=5.5, 12.8 Hz, H-3eq), 2.03 (s, 3H,ANC), 1.85 (t, 1H, J=12.0 Hz, H-ax), 1.45-1.20 (m, 44H, 22CH2), 0.90 (t,6H, J=7.0 Hz, 2CH2CH3).

Example 9

3-O-[Sodium{5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycerol-α-D-galacto-2-nonulopyranosyl)onate]-1,2-di-O-octadecyl-Sn-glycerol

3-O-[Sodium(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl)onate]-1,2-di-O-octadecyl-Sn-glycerol[Japanese Patent Laid-Open Publication No. Hei1-125394] (45 mg) wasreacted by the general procedure according to Example 1 to obtain thetitle compound (44 mg, 66%) as white solid.

¹H-NMR (CD₃OD) δ: 3.02 (dd, 1H, J=5.1, 13.2 Hz, H-3eq), 1.92 (s, 3H,NAc), 1.82 (t, 1H, J=12.5 Hz, H-3ax), 1.38-1.22 (m, 60H, 30CH₂), 0.90(t, 6H, J=6.9 Hz, 2CH₂CH₃).

Example 10

3-O-[Sodium{5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosyl}onate]-2-O-(sodiumoxysulfonyl)-1-O-octadecyl-Sn-glycerol

3-O-[Sodium(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl)onate]-1-O-octadecyl-Sn-glycerol[Japanese Patent Publication No. Hei1-125394] (132 mg) was reacted bythe general procedure according to Example 1 to obtain the titlecompound (125 mg, 53%) as white solid.

¹H-NMR (CD₃OD) δ: 3.07 (dd, 1H, J=5.1, 12.1 Hz, H-3eq), 1.95 (s, 3H,NAc), 1.68 (t, 1H, J=12.1 Hz, H-3ax), 1.36-1.22 (m, 30H, 15CH₂), 0.90(t, 3H, J=7.0 Hz, CH₂CH₃).

Example 11

3-O-[Sodium{5-acetamido-3,5-dideoxy4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-β-D-galacto-2-nonulopyranosyl}onate]-2-O-(sodiumoxysulfonyl)-1-O-octadecyl-Sn-glycerol

3-O-[Sodium(5-acetamido-3,5-dideoxy-D-glycero-β-D-galacto-2-nonulopyranosyl)onate]-1-O-octadecyl-Sn-glycerol[Japanese Patent Publication No. Hei1-125394] (66 mg) was reacted by thegeneral procedure according to Example 1 to obtain the title compound(31 mg, 49%) as white solid.

¹H-NMR (CD₃OD) δ: 2.81 (dd, 1H, J=4.4, 12.1 Hz, H-3eq), 2.03 (s, 3H,NAc), 1.88 (t, 1H, J=11.9 Hz, H-3ax), 1.38-1.17 (m, 30H, 15CH₂), 0.90(t, 3H, J=7.0 Hz, CH₂CH₃).

Example 12

3-O-[Sodium{5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosyl}onate]-1,2-di-O-docosyl-Sn-glycerol

3-O-[Sodium(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl)onate]-1,2-di-O-docosyl-Sn-glycerol[Japanese Patent Publication No. Hei1-125394] (502 mg) was reacted bythe general procedure according to Example 1 to obtain the titlecompound (447 mg, 54%) as white solid.

¹H-NMR (CD₃OD-D₂O, 1:1, 50° C.) δ: 2.96 (br. dd, 1H, H-3eq), 1.97 (s,3H, NAc), 1.83 (br. t, 1H, H-3ax), 1.40-1.21 (m, 76H, 38CH₂), 0.89 (t,6H, J=6.4 Hz, 2CH₂CH₃).

Example 13

3-O-[Sodium{5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-β-D-galacto-2-nonulopyranosyl}onate]-1,2-di-O-docosyl-Sn-glycerol

3-O-[Sodium(5-acetamido-3,5-dideoxy-D-glycero-β-D-galacto-2-nonulopyranosyl)onate]-1,2-di-O-docosyl-Sn-glycerol[Japanese Patent Publication No. Hei1-125394] (44 mg) was reacted by thegeneral procedure according to Example 1 to obtain the title compound(25 mg, 40%) as white solid.

¹H-NMR (CDCl₃-CD₃OD-D₂O, 3:4:2) δ: 2.75 (br. dd, 1H, H-3eq), 2.02 (s,3H, NAc), 1.89 (br. t, 1H, H-3ax), 1.40-1.18 (m, 76H, 38CH₂), 0.89 (t,6H, J=6.6 Hz, 2CH₂CH₃).

Example 14

3-S-[Sodium{5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosyl}onate]-1,2-di-O-tetradecyl-Sn-thioglycerol

3-S-[Sodium(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl)onate]-1,2-di-O-tetradecyl-Sn-thioglycerol[Japanese Patent Laid-Open Publication No. Sho64-52794] (41 mg) wasreacted by the general procedure according to Example 1 to obtain thetitle compound (47 mg, 77%) as white solid.

¹H-NMR (CD₃OD) δ: 3.17 (dd, 1H, J=5.1, 12.1 Hz, H-3eq), 1.92 (s, 3H,NAc), 1.77 (t, 1H, J=11.7 Hz, H-3ax), 1.41-1.23 (m, 44H, 22CH₂), 0.90(t, 6H, J=7.1 Hz, 2CH₂CH₃).

Example 15

3-O-[Methyl(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl)onate]-1,2-di-O-eicosyl-Sn-glycerol

A 3-O-benzyl-1,2-di-O-eicosyl-Sn-glycerol

3-O-Benzyl-Sn-glycerol [Agric. Biol. Chem., 46, 255 (1982)] (300 mg,1.65 mmol), 1-bromo-eicosane (2.38 g, 6.58 mmol) and pulverized sodiumhydroxide (293 mg, 7.33 mmol) were azeotropically heated at reflux inbenzene (10 ml) for 2 days to remove water from the mixture. After thereaction solution was diluted with ether and washed with water, theorganic layer was dried over anhydrous magnesium sulfate and thencondensed in vacuo. The residue was purified by silica gel columnchromatography (100 g of gel, hexane:toluene=3:2) to obtain the titlecompound (929 mg, 76%) as white powder.

¹H-NMR (CDCl₃) δ: 7.34-7.26 (m, 5H, C₆H₅), 4.55 (s, 2H, CH₂Ph), 1.25 (m,68H, 34H₂), 0.88 (t, 6H, J=6.6 Hz, 2CH₂CH₃).

B 1,2-Di-O-eicosyl-Sn-glycerol

3-O-benzyl-1,2-di-O-eicosyl-Sn-glycerol (1.47 g, 1.98 mmol) and 10%palladium-charcoal (200 mg). were stirred in ethyl acetate (30 ml) underthe hydrogen atmosphere at room temperature for 18 h. The reactionmixture was filtered through celite, and the filtrate was concentratedin vacuo to obtain the title compound (934 mg, 72%) as white powder.

¹H-NMR (CDCl₃) δ: 1.25 (m, 68H, 34CH₂), 0.88 (t, 6H, J=6.6 Hz, 2CH₂CH₃).

C3-O-[Methyl(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl)onate]-1,2-di-O-eisosyl-Sn-glycerol

A mixture comprising 1,2-di-O-eicosyl-Sn-glycerol (497 mg, 0.76 mmol),methyl5-acetamido-4,7,8,9-tetra-O-acetyl-2-chloro-2,3,5-trideoxy-D-glycero-β-D-galacto-2-nonulopyranosonate[Chem. Ber., 99, 611 (1966)] (422 mg, 0.83 mmol), mercury (II) cyanide(336 mg, 1.33 mmol), mercury (II) bromide (478 mg, 1.33 mmol) and driedmolecular sieve 4A (1.0 g) in anhydrous chloroform (5.0 ml) was stirredunder the nitrogen atmosphere at room temperature overnight. After thereaction mixture was filtered, the filtrate was concentrated in vacuo,and purified by intermediary pressure silica gel column chromatography(125 g of gel, toluene:ethyl acetate=3:2) to obtain the title compound(390 mg, 46%) as white power.

¹H-NMR (CDCl₃) δ: 3.79 (s, 3H, OCH₃), 2.60 (dd, 1H, J=4.8, 12.8 Hz,H-3eq.), 2.14, 2.13, 2.06, 2.04, 1.88 (5s, 15H, 5Ac), 1.98 (t, 1H,J=12.8 Hz, H-3ax.), 1.25 (m, 68H, 34CH₂), 0.88 (t, 6H, J=7.0 Hz,2CH₂CH₃).

Example 16

3-O-[(5-Acetamido-3,5-dideoxy-D-glycero-α-D-galacto-nonulopyranosonicacid)-2-yl]-1,2-di-O-eicosyl-Sn-glycerol

3-O-[Methyl(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl)onate]-1,2-di-O-eicosyl-Sn-glycerol(455 mg, 0.40 mmol)and sodium methoxide was stirred in a mixture (13 ml)of THF:methanol (1:1) overnight. To this reaction mixture was added 3Nsodium hydroxide (380 μl), and the mixture was stirred at 60° C. for 1h. After the reaction mixture was concentrated in vacua, the residue wasdissolved in ethanol and water, and the solution was adjusted to pH 2with formic acid. Precipitates were collected by filtration, washedsuccessively with water, methanol and ether, and dried in vacua toobtain the title compound (365 mg, 96%)as white powder.

¹H-NMR (CDCl₃-CD₃OD, 1:1) δ: 2.79 (dd, 1H, J=3.9, 12.3 Hz, H-3eq.), 2.03(s, 3H, NAc), 1.69 (t, 1H, J=11.5 Hz, H-3ax.), 1.43-1.20 (m, 68H,34CH₂), 0.89 (t, 6H, J=7.0 Hz, 2CH₂CH₃).

Example 17

3-O-[Sodium{5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosyl)onate]-1,2-di-O-eicosyl-Sn-glycerol

3-O-[(5-Acetamido-3,5-dideoxy-D-glycero-α-D-galacto-nonulopyranosonicacid)-2-yl]-1,2-di-O-eicosyl-Sn-glycerol (241 mg) was reacted by thegeneral procedure according to Example 1 to obtain the title compound(236 mg, 67%) as white solid.

¹H-NMR (CD₃OD) δ: 3.02 (dd, 1H, J=4.4, 12.4 Hz, H-3eq.), 1.92 (s, 3H,NAc), 1.80 (t, 1H, J=11.0 Hz, H-3ax.), 1.44-1.13 (m, 68H, 34CH₂), 0.909(t, 6H, J=6.8 Hz, 2CH₂CH₃).

Example 18

3-O-[Methyl(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl}onate]-1,2-di-O-tetracosyl-Sn-glycerol

A 3-O-Benzyl-1,2-di-O-tetracosyl-Sn-glycerol

3-O-Benzyl-Sn-glycerol [Agric. Biol. Chem., 46, 255 (1982)] and1-bromo-tetracosane [J. Am. Chem. Soc., 115, 3840 (1993)] were reactedby the general procedure according to Example 1 to obtain the titlecompound (236 mg, 67%) as white solid.

¹H-NMR (CDCl₃) δ: 7.34-7.26 (m, 5H, C₆H₅), 4.55 (s, 2H, CH₂Ph), 1.25 (m,84H, 42CH₂), 0.88 (t, 6H, J=6.6 Hz, 2CH₂CH₃).

B 1,2-Di-O-tetracosyl-Sn-glycerol

3-O-Benzyl-1,2-di-O-tetracosyl-Sn-glycerol (1.47 g) was reacted in amixture (30 ml) of toluene-ethyl acetate (1:1) by the general procedureaccording to 15-B to obtain the title compound (0.80 g, 66%) as whitesolid.

¹H-NMR (CDCl₃) δ: 1.25 (m, 84H, 42CH₂), 0.88 (t, 6H, J=6.6 Hz, 2CH₂CH₃).

C3-O-[Methyl(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl}onate]-1,2-di-O-tetracosyl-Sn-glycerol

1,2-Di-O-tetracosyl-Sn-glycerol (497 mg, 0.76 mmol) and methyl5-acetamido-4,7,8,9-tetra-O-acetyl-2-chloro-2,3,5-trideoxy-D-glycero-β-D-galacto-2-nonulopyranosonate[Chem. Ber., 99, 611 (1966)] (422 mg, 0.83 mmol) were reacted by thegeneral procedure according to 15-C to obtain the title compound (224mg, 14%) as white solid.

¹H-NMR (CDCl₃) δ: 3.79 (s, 3H, OCH₃), 2.60 (dd, 1H, J=4.8, 12.8 Hz,H-3eq), 2.14, 2.13, 2.04, 2.03, 1.88 (5s, 15H, 5Ac), 1.98 (t, 1H, J=12.8Hz, H-3ax), 1.25 (m, 84H, 42CH₂), 0.88 (t, 6H, J=6.6 Hz, 2CH₂CH₃).

Example 19

3-O-[(5-Acetamido-3,5-dideoxy-D-glycero-α-D-galacto-nonulopyranosonicacid)-2-yl]-1,2-di-O-tetracosyl-Sn-glycerol

3-O-[Methyl(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl}onate]-1,2-di-O-tetracosyl-Sn-glycerol(240 mg) was reacted by the general procedure according to Example 16 toobtain the title compound (160 mg, 79%) as white solid.

¹H-NMR (CDCl₃-CD₃OD, 1:1) δ: 2.81 (dd, 1H, J=3.7, 12.1 Hz, H-3eq), 2.03(s, 3H, NAc), 1.66 (t, 1H, J=11.3 Hz, H-3ax), 1.38-1.16 (m, 84H, 42CH₂),0.89 (t, 6H, J=6.8 Hz, 2CH₂CH₃).

Example 20

3-O-[Sodium{5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosyl}onate]-1,2-di-O-tetracosyl-Sn-glycerol

3-O-[(5-Acetamido-3,5-dideoxy-D-glycero-α-D-galacto-nonulopyranosonicacid) reacted by the general procedure according to Example 1 to obtainthe title compound (21 mg, 19%) as white solid.

¹H-NMR (CD₃OD-D₂O, 1:1, 50° C.) δ: 2.96 (br. dd, 1H, H-3eq), 2.01 (s,3H, NAc), 1.81 (br. t, 1H, H-3ax), 1.36-1.10 (m, 84H, 42CH₂), 0.89 (t,6H, J=6.4 Hz, 2CH₂CH₃).

Example 21

3-O-[Methyl(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl)onate]-1,2-di-O-hexacosyl-Sn-glycerol

A 3-O-Benzyl-1,2-di-O-hexacosyl-Sn-glycerol

3-O-Benzyl-Sn-glycerol [Agric. Biol. Chem., 40, 391 (1976)] (242 mg) and1-bromohexacosane [Agric. Biol. Chem., 46, 255 (1982)] (1.30 g) werereacted by the general procedure according to Example 15-A. Then, thecompound thus obtained was, without purification, subjected to thedebenzylation by the general procedure according to Example 15-B toobtain the title compound (615 mg, 57%) as white solid.

¹H-NMR (CDCl₃) δ: 1.25 (m, 92H, 46CH₂), 0.88 (t, 6H, J=6.6 Hz, 2CH₂CH₃).

B3-O-[Methyl(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl)onate]-1,2-di-O-hexacosyl-Sn-glycerol

1,2-Di-O-hexacosyl-Sn-glycerol (610 mg, 0.74 mmol) and methyl5-acetamido-4,7,8,9-tetra-O-acetyl-2-chloro-2,3,5-trideoxy-D-glycero-β-D-galacto-2-nonulopyranosonate[Chem. Ber., 99, 611 (1966)] (454 mg, 0.89 mmol) were reacted by thegeneral procedure according to Example 15-C to obtain the title compound(76 mg, 8%) as white solid.

¹H-NMR (CDCl₃) δ: 3.79 (s, 3H, OCH₃), 2.60 (dd, 1H, J=4.8, 12.8 Hz,H-3eq), 2.14, 2.13, 2.04, 2.02, 1.88 (5s, 15H, 5Ac), 1.97 (t, 1H, J=12.8Hz, H-3ax), 1.25 (m, 92H, 46CH₂), 0.88 (t, 6H, J=7.0 Hz, 2CH₂CH₃).

Example 22

3-O-[(5-Acetamido-3,5-dideoxy-D-glycero-α-D-galacto-nonulopyranosonicacid)-2-yl]-1,2-di-O-hexacosyl-Sn-glycerol

3-O-[Methyl(5-acetamido4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl)onate]-1,2-di-O-hexacosyl-Sn-glycerol(76 mg) was reacted by the general procedure according to Example 16 toobtain the title compound (51 mg, 78%) as white solid.

¹H-NMR (CDCl₃-CD₃OD, 1:1, 60° C.) δ: 2.76 (dd, 1H, J=4.4, 12.8 Hz,H-3eq), 2.02 (s, 3H, NAc), 1.75 (t, 1H, J=11.7 Hz, H-3ax), 1.42-1.14 (m,92H, 46CH₂), 0.89 (t, 6H, J=6.8 Hz, 2CH₂CH₃).

Example 23

3-O-[Sodium{5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosyl}onate]-1,2-di-O-hexacosyl-Sn-glycerol

3-O-[(5-Acetamido-3,5-dideoxy-D-glycero-α-D-galacto-nonulopyranosonicacid)-2-yl]-1,2-di-O-hexacosyl-Sn-glycerol (47 mg) was reacted by thegeneral procedure according to Example 1 to obtain the title compound(29 mg, 45%) as white solid.

¹H-NMR (CD₃OD-D₂O, 1:1, 60° C.) δ: 2.96 (br. dd, 1H, H-3eq), 1.96 (s,3H, NAc), 1.79 (br. t, 1H, H-3ax), 1.42-1.14 (m, 92H, 46CH₂), 0.88 (br.t, 6H, J=6.2 Hz, 2CH₂CH₃).

Example 24

3β-[Sodium{5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosyl}onate]-5-cholestene

3β-[(5-Acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosonicacid)-2-yl]-5-cholestene [Japanese Patent Laid-Open Publication No. Sho61-243096] (387 mg) was reacted by the general procedure according toExample 1 to obtain the title compound (232 mg, 37%) as white solid.

¹H-NMR (CD₃OD-D₂O, 1:1) δ: 2.92 (dd, 1H, J=4.5, 11.5 Hz, H-3eq), 1.96(s, 3H, NAc), 0.99 (s, 3H, CH₃-19 chole), 0.68 (s, 3H, CH₃-18 chole).

Example 25

Sodium[oleyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosid]onate

A Sodium(oleyl5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosid)onate

To a solution of methyl (oleyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosid)onate[Japanese Patent Laid-Open Publication No. Sho 63-264493](115 mg, 0.154mmol) dissolved in methanol (1.5 ml) was added 3N sodium hydroxide (0.30ml), and the mixture was stirred at room temperature for 16 h. Thereaction solution was evaporated to dryness in vacua, and the residuewas purified by gel chromatography (Sephadex LH-20, 180 ml, methanol) toobtain the title compound (78 mg, 87%) as white solid.

¹H-NMR (CD₃OD) δ: 5.34 (m, 2H, CH═CH), 2.81 (dd, 1H, J=4.0, 12.1 Hz,H-3eq), 2.00 (s, 3H, NAc), 1.59 (t, 1H, J=11.7 Hz, H-3ax), 1.42-1.23 (m,20H, 10CH₂), 0.90 (t, 3H, J=7.0 Hz, CH₂CH₃).

B Sodium[oleyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosid]onate

Sodium(oleyl5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosid)onate(62 mg) was reacted by the general procedure according to Example 1 toobtain the title compound (86 mg, 82%) as white solid.

¹H-NMR (CD₃OD) δ: 3.03 (dd, 1H, J=3.7, 12.3 Hz, H-3eq), 2.19 (m, 4H,CH₂CH═CHCH₂), 1.92 (s, 3H, NAc), 1.73 (t, 1H, J=12.1 Hz, H-3ax),1.42-1.22 (m, 20H, 10CH₂), 0.90 (t, 3H, J=6.8 Hz, CH₂CH₃).

Example 26

Sodium[octadecyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosid)onate

A Methyl(octadecyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosid)onate

To a solution of methyl(oleyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosid)onate[Japanese Patent Laid-Open Publication No. Sho 63-264493] (111 mg, 0.149mmol) dissolved in ethanol (1.5 ml) was added 10% palladium-charcoal (16mg) was added, and the mixture was stirred under the hydrogen atmosphereat room temperature for 18 h. The reaction solution was filtered throughcelite to obtain the title compound (107 mg, 96%) as white solid.

¹H-NMR (CDCl₃) δ: 3.79 (s, 3H, COOCH₃), 2.58 (dd, 1H, J=4.8, 12.8 Hz,H-3eq), 2.15, 2.14, 2.04, 2.03, 1.88 (5s, 15H, 5Ac), 1.95 (t, 1H, J=12.6Hz, H-3ax), 1.38-1.19 (m, 30H, 15CH₂), 0.88 (t, 3H, J=7.0 Hz, CH₂CH₃).

B Sodium(octadecyl5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosid)onate

Methyl(octadecyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosid)onate(107 mg) was reacted by the general procedure according to Example 25-Ato obtain the titled compound (70 mg, 83%) as white solid.

¹H-NMR (CD₃OD) δ: 2.81 (dd, 1H, J=3.8, 12.3 Hz, H-3eq), 2.00 (s, 3H,NAc), 1.59 (t, 1H, J=11.9 Hz, H-3ax), 1.38-1.22 (m, 30H, 15CH₂), 0.90(t, 3H, J=6.8 Hz, CH₂CH₃).

C Sodium[octadecyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosid]onate

Sodium(octadecyl5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulo-pyranosid)onate(72 mg) was reacted by the general procedure according to Example 1 toobtain the title compound (109 mg, 92%) as white solid.

¹H-NMR (CD₃OD) δ: 3.02 (dd, 1H, J=3.7, 11.4 Hz, H-3eq), 1.92 (s, 3H,NAc), 1.73 (t, 1H, J=11.7 Hz, H-3ax), 1.40-1.31 (m, 30H, 15CH₂), 0.90(t, 3H, J=7.0 Hz, CH₂CH₃).

Example 27

Methyl[2,2-bis(docosyloxymethyl)propyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosid]onate

A 2,2-bis(docosyloxymethyl)propanol

1,1,1-tris(hydroxymethyl)ethane (1.0 g, 8.32 mmol) and sodium hydride(732 mg, 18.3 mmol) were stirred in dehydrated dimethylformamide (30 ml)at room temperature for 15 mm. Then, the reaction solution was cooled inice, and docosyl bromide (7.1 g, 18.3 mmol) and benzene (10 ml) wereadded thereto, and the resulting mixture was stirred at room temperaturefor 16 h. The reaction solution was concentrated in vacuo, and theresidue was suspended in chloroform, and washed with 2HCl. The organiclayer was dried over anhydrous magnesium sulfate, and concentrated invacuo. The residue was purified by silica gel column chromatography (130g of gel, toluene/ethyl acetate, 19:1) to obtain the title compound(42.2 g, 38%) as white powder.

B Methyl[2,2-bis(docosyloxymethyl)propyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosid]onate

2,2-Bis(docosyloxymethyl)propanol (1.20 g, 1.63 mmol) and methyl5-acetamido-4,7,8,9-tetra-O-acetyl-2-chloro-2,3,5-trideoxy-D-glycero-β-D-galacto-2-nonulopyranosonate [Chem. Ber., 99, 611 (1966)] (913 mg, 1.79 mmol) werereacted by the general procedure according to Example 15-C to obtain thetitle compound (786 mg, 40%) as white solid.

¹H-NMR (CDCl₃) δ: 3.78 (s, 3H, COOCH₃), 2.57 (dd, 1H, J=4.4, 12.8 Hz,H-3eq.), 2.13, 2.12, 2.04, 2.02, 1.88 (5s, 15H, 5Ac), 1.95 (t, 1H,J=12.5 Hz, H-3ax.), 1.25 (m, 76H, 38CH₂), 0.90 (s, 3H, CCH₃), 0.88 (t,6H, J=7.0 Hz, 2CH₂CH₃).

Example 28

2,2-Bis(docosyloxymethyl)propyl5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosidonicacid:

Methyl[2,2-bis(docosyloxymethyl)propyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosid]onate(786 mg) was reacted by the general procedure according to Example 16 toobtain the title compound (599 mg, 90%) as white solid.

¹H-NMR (CDCl₃-CD₃OD, 1:1, 60° C.) δ: 2.79 (dd, 1H, J=4.8, 12.8 Hz,H-3eq), 2.02 (s, 3H, NAc), 1.75 (t, 1H, J=11.9 Hz, H-3ax), 1.36-1.22 (m,76H, 38CH₂), 0.92 (s, 3H, CCH₃), 0.89 (t, 6H, J=6.8 Hz, 2CH₂CH₃).

Example 29

Sodium[2,2-bis(docosyloxymethyl)propyl5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosid]onate

2,2-Bis(docosyloxymethyl)propyl5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosidonic acid(94 mg) was reacted by the general procedure according to Example 1 toobtain the title compound (63 mg, 47%) as white solid. (599 mg, 90%) aswhite solid.

¹H-NMR (CD₃OD-D₂O, 1:1, 60° C.) δ: 2.93 (br. dd, 1H, H-3eq), 1.96 (s,3H, NAc), 1.80 (br. t, 1H, H-3ax), 1.43-1.16 (m, 76H, 38CH₂), 0.92 (s,3H, CCH₃), 0.89 (t, 6H, J=6.6 Hz, 2CH₂CH₃).

Example 30

Methyl[2,2-bis(eicosyloxymethyl)propyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosid]onate

A 2,2-Bis(eicosyloxymethyl)propanol

1,1,1-tris(hydroxymethyl)ethane (0.50 g, 4.16 mmol) and eicosyl bromide(3.30 g, 9.15 mmol) were reacted by the general procedure according toExample 27-A to obtain the title compound (1.43 g, 51%) as white solid.

¹H-NMR (CDCl₃) δ: 3.57 (d, 2H, J=5.9 Hz, CH₂OH), 1.26 (m, 68H, 34CH₂),0.88 (t, 6H, J=7.0 Hz, 2CH₂CH₃), 0.85 (s, 3H, CCH₃).

B Methyl[2,2-bis(eicosyloxymethyl)propyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosid]onate

2,2-bis(eicosyloxymethyl)propanol (267 mg, 0.392 mmol) and methyl5-acetamido-4,7,8,9-tetra-O-acetyl-2-chloro-2,3,5-trideoxy-D-glycero-β-D-galacto-2-nonulopyranosonate[Chem. Ber., 99, 611 (1966)] (200 mg, 0.392 mmol) were reacted by thegeneral procedure according to Example 15-C to obtain the title compound(175 mg, 39%) as white solid.

¹H-NMR (CDCl₃) δ: 3.78 (s, 3H, COOCH₃), 2.58 (dd, 1H, J=4.8, 12.8 Hz,H-3eq.), 2.13, 2.13, 2.04, 2.02, 1.89 (5s, 15H, 5Ac), 1.95 (dd, 1H,J=12.5, 12.8 Hz, H-3ax.), 1.25 (m, 68H, 34CH₂), 0.90 (s, 3H, CCH₃), 0.88(t, 6H, J=7.0 Hz, 2CH₂CH₃).

Example 31

2,2-bis(eicosyloxymethyl)propyl5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosidonicacid:

Methyl[2,2-bis(eicosyloxymethyl)propyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosid]onate(174 mg) was reacted by the general procedure according to Example 16 toobtain the title compound (127 mg, 87%) as white solid.

¹H-NMR (CDCl₃-CD₃OD, 1:1) δ: 2.75 (dd, 1H, J=4.6, 12.6 Hz, H-3eq), 2.03(s, 3H, NAc), 1.75 (t, 1H, J=11.7 Hz, H-3ax), 1.43-1.19 (m, 68H, 34CH₂),0.92 (s, 3H, CCH₃), 0.89 (t, 6H, J=7.0 Hz, 2CH₂CH₃).

Example 32

Sodium[2,2-bis(eicosyloxymethyl)propyl5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosid)onate

2,2-bis(eicosyloxymethyl)propyl5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosidonic acid(103 mg) acid was reacted by the general procedure according to Example1 to obtain the title compound (96 mg, 64%) as white solid.

¹H-NMR (CD₃OD-D₂O, 1:1, 60° C.) δ: 2.96 (br. dd, 1H, H-3eq), 1.98 (s,3H, NAc), 1.78 (t, 1H, J=12.6 Hz, H-3ax), 1.37-1.17 (m, 68H, 34CH₂),0.94 (s, 3H, CCH₃), 0.90 (t, 6H, J=6.8 Hz, 2CH₂CH₃).

Example 33

Methyl(3,5-didocosyloxyphenyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosid)onate

A 3,5-didocosyloxyphenol

After phloroglucinol (2.0 g, 15.9 mmol) and sodium hydride (1.59 g, 39.7mmol) were stirred in dehydrated dimethylformamide (30 ml) at roomtemperature for 15 min, docosyl bromide (13.0 mg, 33.3 mmol) and benzene(30 ml) were added thereto, and the resulting mixture was stirred at 40°C. for 2 days. The reaction solution was diluted with chloroform, washedwith 2N hydrochloric acid, and the organic layer was dried overanhydrous magnesium sulfate. The solvent was concentrated in vacuo, andthe residue was purified by silica gel column chromatography (90 g ofthe gel, hexane/ethyl acetate, 6:1) to obtain the title compound (2.40g, 20%) as light yellow powder.

¹H-NMR (CDCl₃) δ: 6.06 (d, 1H, J=2.2 Hz, H-4 phenyl), 5.99 (2d, 2H,J=2.2 Hz, H-2,6 phenyl), 3.89 (t, 4H, J=6.8 Hz, 2OCH₂), 1.39-1.17 (m,72H, 36CH₂), 0.88 (t, 6H, J=6.8 Hz, 2CH₂CH₃).

B Methyl(3,5-didocosyloxyphenyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosid)onate

3,5-Didocosyloxyphenol (1.20 g, 1.61 mmol) and sodium hydride (77.5 g,1.94 mmol) were stirred in dimethylformamide:benzene (1:1, 40 ml) atroom temperature for 20 min. Then, to this mixture was added methyl5-acetamido-4,7,8,9-tetra-O-acetyl-2-chloro-2,3,5-trideoxy-D-glycero-β-D-galacto-2-nonulopyranosonate[Chem. Ber., 99, 611 (1966)] (823 mg, 1.61 mmol), and the resultingmixture was stirred at room temperature for 3 h. After the reactionsolution was diluted with chloroform, and washed with 2N-hydrochloricacid and, the organic layer was dried over anhydrous magnesium sulfate.The solvent was condensed in vacuo, and the residue was purified bysilica gel column chromatography (90 g of the gel, toluene/ethylacetate, 2:3) to obtain the title compound (386 mg, 20%) light yellowpowder.

¹H-NMR (CDCl₃) δ: 6.28 (2d, 2H, J=1.8 Hz, H-2,6phenyl), 6.22 (d, 1H,J=1.8 Hz, H-4phenyl), 3.78 (s, 3H, COOCH₃), 2.58 (dd, 1H, J=4.8, 12.8Hz, H-3eq.), 2.14, 2.10, 2.03, 2.01, 1.90 (5s, 15H, 5Ac), 1.26 (m, 76H,38CH₂), 0.88 (t, 6H, J=6.6 Hz, 2CH₂CH₃).

Example 34

3,5-Didocosyloxyphenyl5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyrasidonic acid:

Methyl(3,5-didocosyloxyphenyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosid)onate(385 mg) was reacted by the general procedure according to Example 16 toobtain the title compound (163 mg, 50%) as light yellow solid.

¹H-NMR (CDCl₃-CD₃OD, 1:1, 60° C.) δ: 6.42 (2d, 2H, J=2.2 Hz,H-2,6phenyl), 6.20 (d, 1H, J=2.2 Hz, H-4phenyl), 2.86 (dd, 1H, J=4.2,12.6 Hz, H-3eq), 2.02 (s, 3H, NAc), 1.95 (t, 1H, J=11.9 Hz, H-3ax),1.40-1.23 (m, 72H, 36CH₂), 0.89 (t, 6H, J=7.0 Hz, 2CH₂CH₃).

Example 35

Sodium[3,5-Didocosyloxyphenyl-5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-β-D-galacto-2-nonulopyranosid)onate

3,5-Didocosyloxyphenyl5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulo yranosidonic acid(16 mg) was reacted by the general procedure according to Example 1 toobtain the title compound (11 mg, 47%) as white solid.

¹H-NMR (CD₃OD-D₂O, 1:1, 50° C.) δ: 6.42 (br. s, 2H, H-2,6phenyl), 6.17(br. s, 1H, H-4phenyl), 2.93 (br. dd, 1H, H-3eq), 2.00 (s, 3H, NAc),1.38-1.18 (m, 72H, 36CH₂), 0.90 (t, 6H, J=7.0 Hz, 2CH₂CH₃).

Example 36

3-O-[Methyl(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl)onate]-1,2-di-O-docosanoyl-Sn-glycerol

A 3-O-Benzyl-1,2-di-O-docosanoyl-Sn-glycerol

3-O-Benzyl-Sn-glycerol [Agric. Biol. Chem., 40, 391 (1976)] (0.67 g,3.68 mmol), behenic acid (3.76 g, 11.04 mmol), dicyclohexylcarbodiimide(2.28 g, 11.04 mmol) and 4-dimethylaminopyridine (0.13 g, 1.06 mmol)were stirred in pyridine (37 ml)at room temperature for 22 h. Thereaction solution was filtered, and evaporated to dryness in vacuo. Theresidue was purified by silica gel column chromatography (using 90 g ofgel, hexane/ethyl acetate, 9:1) to obtain the title compound (1.55 g,51%) as light yellow powder.

¹H-NMR (CDCl₃) δ: 7.30 (m, 5H, C₆H₅), 5.24 (m, 1H, H-2), 4.56 (d, 1H,J=12.1 Hz, C₆H₅CH), 4.52 (d, 1H, J=12.1 Hz, C₆H₅CH), 1.25 (m, 72H,36CH₂), 0.88 (t, 6H, J=7.0 Hz, 2CH₂CH₃).

B 1,2-Di-O-docosanoyl-Sn-glycerol

3-O-Benzyl-1,2-di-O-docosanoyl-Sn-glycerol (1.25 g) was reacted by thegeneral procedure according to Example 15-B to obtain the title compound(0.98 g, 88%) as white solid.

¹H-NMR (CDCl₃) δ: 5.08 (m, 1H, H-2), 4.32 (dd, 1H, J=4.6, 11.9 Hz, H-3),4.24 (dd, 1H, J=5.7, 11.9 Hz, H-3), 3.73 (m, 2H, CH₂-1), 1.25 (m, 72H,36CH₂), 0.88 (t, 6H, J=7.0 Hz, 2CH₂CH₃).

C3-O-[Methyl(5-acetamido-4,7,8,9-tetra-O-levulinoyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl)onate]-1,2-di-O-docosanoyl-Sn-glycerol

Methyl 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-2-nonulopyranosonate[Chem. Ber., 99, 611 (1966)] (3.23 g, 10.0 mmol),4-dimethylaminopyridine (0.61 g, 0.50 mmol), levulinic acid (12.3 ml,120 mmol) and dicyclohexylcarbodiimide (24.76 g, 120 mmol) were stirredin pyridine (30 ml) at room temperature for two days. The reactionsolution was filtered through celite, and the filtrate was evaporated todryness in vacuo. The residue was purified by silica gel columnchromatography (with 170 g of gel, chloroform/methanol, 24:1) to obtainthe penta-levulinoyl derivative (4.62 g, 65%) of the title compound.Then the penta-levulinoyl derivative (2.50 g, 3.07 mmol) was dissolvedin acetyl chloride (30 ml), saturated with hydrogen chloride gas at 0°C., left at standing at 2° C. for five days. The reaction solution wasevaporated to dryness in vacuo to obtain the chloride derivative (2.15g, 95%) of the title compound. Then, the chloride derivative (1.22 g,1.66 mmol) and 1,2-di-O-docosanoyl-Sn-glycerol (0.89 g, 1.21 mmol) werereacted by the general procedure according to Example 15-C to obtain thetitle compound (64 mg, 4%) as white solid.

¹H-NMR (CDCl₃) δ: 5.06 (dt, 1H, J=4.6, 11.7 Hz, H-4), 3.81 (s, 3H,COOCH₃), 2.19 (6H), 2.18, 2.17 (3s, 12H, 3CH₃CO), 1.89 (s, 3H, NAc),1.85 (t, 1H, J=12.5 Hz, H-3ax), 1.25 (m, 72H, 36CH₂), 0.88 (t, 6H, J=7.0Hz, 2CH₂CH₃).

Example 37

3-O-[(5-Acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosonicacid-2-yl]-1,2-di-O-docosanoyl-Sn-glycerol

3-O-[Methyl(5-acetamido-4,7,8,9-tetra-O-levulinoyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl)onate]-1,2-di-O-docosanoyl-Sn-glycerol(52 mg, 0.036 mmol) and anhydrous lithium iodide (49 mg, 0.366 mmol)were stirred in dehydrated pyridine (1.8 ml) at 80° C. for 6 h. Thereaction solution was directly purified by gel chromatography (usingSephadex LH-20, 100 ml, chloroform/methanol, 1:1) to obtain lithium saltof title compound (34.6 mg, 67%) as white solid. The lithium salt (34.6mg, 0.0242 mmol) was dissolved in methanol (0.5 ml) and chloroform (0.5ml), added with hydrazine acetate (48 mg, 0.524 mmol), and the mixturewas stirred at room temperature for 10 min. The reaction solution wasadjusted to pH 4 with 0.1N-hydrochloric acid, and purified by gelchromatography (Sephadex LH-20, 75 ml, chloroform/methanol, 1:1) toobtain the title compound (23 mg, 92%) as white solid.

¹H-NMR (CDCl₃-CD₃OD, 1:1, 40° C.) δ: 2.76 (dd, 1H, J=4.2, 12.6 Hz,H-3eq), 2.03 (s, 3H, NAc), 1.74 (t, 1H, J=11.9 Hz, H-3ax), 1.28 (m, 72H,36CH₂), 0.89 (t, 6H, J=7.0 Hz, 2CH₂CH₃).

Example 38

3-O-[Sodium{5-acetamido-3,5-dideoxy-4,7,8,9-tretra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosyl}onate]-1,2-di-O-docosanoyl-Sn-glycerol

3-O-[(5-Acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosonicacid)-2-yd]-1,2-di-O-docosanoyl-Sn-glycerol (12 mg) was reacted by thegeneral procedure according to Example 1 to obtain the title compound (9mg, 53%) as white solid.

¹H-MR (CD3OD-D2O, 1:1, 50° C.) δ: 2.94 (BR. DD, 1H, H-3eq), 1.95 (s, 3H,ANC), 1.71 (t, 1H, J=11.0 Hz, H-ax), 1.26 (m, 72H, 36CH2), 0.87 (t, 6H,J=6.8 Hz, 2CH2CH3).

Example 39

3-O-[Methyl(4,5,7,8,9-penta-O-acetyl-3-deoxy-D-glycero-α-D-galacto-2-nonulopyranosyl)onate]-1,2-di-O-docosyl-Sn-glycerol

1,2-Di-O-docosyl-Sn-glycerol [Japanese Patent Laid-Open Publication No.Hei 1-125394] (467 mg) and methyl4,5,7,8,9-penta-O-acetyl-2-chloro-2,3-dideoxy-D-glycero-β-D-galacto-2-nonulopyranosonate[Chem. Pharm. Bull., 39, 3140 (1991)] (281 mg) were reacted by thegeneral procedure according to Example 15-C to obtain the title compound(313 mg, 48%) as white solid.

¹H-NMR (CDCl₃) δ: 3.80 (s, 3H, OCH₃, 2.68 (dd, 1H, J=4.0, 12.8 Hz,H-3eq), 2.15, 2.09, 2.04, 2.00, 2.00 (5s, 15H, 5Ac), 1.25 (m, 76H,38CH₂), 0.88 (t, 6H, J=6.6 Hz, 2CH₂CH₃).

Example 40

3-O-[(3-Deoxy-D-glycero-α-D-galacto-2-nonulopyranosonic acidacid)-2-yl]-1,2-di-O-docosyl-Sn-glycerol

3-O-[Methyl(4,5,7,8,9-penta-O-acetyl-3-deoxy-D-glycero-α-D-galacto-2-nonulopyranosyl)onate]-1,2-di-O-docosyl-Sn-glycerol(313 mg) was reacted by the general procedure according to Example 16 toobtain the title compound (186 mg, 75%) as white solid.

¹H-NMR (CDCl₃-CD₃OD, 1:1, 60° C.) δ: 2.68 (dd, 1H, J=4.6, 12.6 Hz,H-3eq), 1.73 (t, 1H, J=12.1 Hz, H-3ax), 1.29 (m, 76H, 38CH₂), 0.89 (t,6H, J=7.0 Hz, 2CH₂CH₃).

Example 41

3-O-[Sodium{3-deoxy-4,5,7,8,9-penta-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosyl}onate]-1,2-di-O-docosyl-Sn-glycerol

3-O-[(3-Deoxy-D-glycero-α-D-galacto-2-nonulopyranosonicacid)-2-yl]-1,2-di-O-docosyl-Sn-glycerol (97 mg) was reacted by thegeneral procedure according to Example 1 to obtain the title compound(15 mg, 10%) as white solid.

¹H-NMR (CD₃OD-D₂O, 1:1, 50° C.) δ: 2.89 (br. dd, 1H, H-3eq), 1.81 (br.t, 1H, H-3ax), 1.26 (m, 76H, 38CH₂), 0.86 (t, 6H, J=7.0 Hz, 2CH₂CH₃).

Example 42

1-O-[Methyl(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl)onate]-L-2,3-di-O-docosyl-Sn-glycerol

A L-1-O-Benyl-2,3-di-O-isopropylidene-Sn-glycerol

After L-2,3-di-O-isopropylidene-Sn-glycerol (5.00 g, 37.8 mmol) and 60%sodium hydride (3.03 g, 75.8 mmol) were stirred in dehydrateddimethylformamide (110 ml) at room temperature for 10 min, benzylbromide (6.7 ml, 56.7 mmol) was added, and the resulting mixture wasstirred at room temperature for 3 h. The reaction solution was dilutedwith ether, washed successively with water and saturated sodium chloridesolution, dried over anhydrous magnesium sulfate, and concentrated invacuo. The residue was distilled under reduced pressure (110° C./3 torr)to obtain the benzyl derivative (6.65 g, 80%). Then, to a solution ofthis product (6.65 g, 29.9 mmol) dissolved in dichloromethane (30 ml),methanol (9 ml) and water (3 ml) was added trifluoroacetic acid (3 ml),and the mixture was stirred at room temperature for 3 h. After thereaction solution was neutralized with sodium hydroxide and condensed invacuo, the residue was purified by distillation under reduced pressureto obtain the title compound (4.71 g, 86%).

¹H-NMR (CDCl₃) δ: 7.32 (m, 5H, C₆H₅), 4.56 (s, 2H, CH₂Ph).

B L-1-O-Benzyl-2,3-di-O-docosyl-Sn-glycerol

L-1-O-Benzyl-Sn-glycerol (1.90 g) and 1-bromo docosane (9.70 g) werereacted by the general procedure according to Example 15-A to obtain thetitle compound (7.0 g, 85%).

¹H-NMR (CDCl₃) δ: 7.30 (m, 5H, C₆H₅), 4.55 (s, 2H, CH₂Ph), 1.25 (m, 76H,38CH₂), 0.88 (t, 6H, J=6.6 Hz, 2CH₂CH₃).

C L-2,3-Di-O-docosyl-Sn-glycerol

L-1-O-Benzyl-2,3-di-O-docosyl-Sn-glycerol (1.40 g) was reacted by thegeneral procedure according to Example 15-B to obtain the title compound(0.99 g, 79%) as white solid.

¹H-NMR (CDCl₃) δ: 1.25 (m, 76H, 38CH₂), 0.88 (t, 6H, J=6.6 Hz, 2CH₂CH₃).

D3-O-[Methyl(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl)-onate]-L-2,3-di-O-docosyl-Sn-glycerol

L-2,3-Di-O-docosyl-Sn-glycerol (990 mg) and methyl5-acetamido-4,7,8,9-tetra-O-acetyl-2-chloro-2,3,5-trideoxy-D-glycero-β-D-galacto-2-nonulopyranosonate[Chem. Ber., 99, 611 (1966)] (850 mg) were reacted by the generalprocedure according to Example 15-C to obtain the title compound (662mg, 40%) as white solid.

¹H-NMR (CDCl₃) δ: 3.79 (s, 3H, OCH₃), 2.60 (dd, 1H, J=4.8, 12.8 Hz,H-3eq.), 2.15, 2.09, 2.04, 2.00, 2.00 (5s, 15H, 5Ac), 1.96 (t, 1H,J=12.8 Hz, H-3ax.), 1.25 (m, 76H, 38CH₂), 0.88 (t, 6H, J=6.6 Hz,2CH₂CH₃).

Example 43

1-O-[(5-Acetamido-3,5-dideoxy-D-glycero-α-D-galacto-nonulopyranosonicacid)-2-yl]-L-2,3-di-O-docosyl-Sn-glycerol

1-O-[Methyl(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl)onate]-L-2,3-di-O-docosyl-Sn-glycerol(632 mg) was reacted by the general procedure according to Example 16 toobtain the title compound (500 mg, 92%) as white solid.

¹H-NMR (CDCl₃-CD₃OD, 1:1, 40° C.) δ: 2.77 (dd, 1H, J=4.6, 13.4 Hz,H-3eq.), 2.02 (s, 3H, NAc), 1.74 (t, 1H, J=11.9 Hz, H-3ax.), 1.28 (m,76H, 38CH₂), 0.89 (t, 6H, J=6.8 Hz, 2CH₂CH₃).

Example 44

1-O-[Sodium{5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosyl}onate]-L-2,3-di-O-docosyl-Sn-glycerol

1-O-[(5-Acetamido-3,5-dideoxy-D-glycero-α-D-galacto-nonulopyranosonicacid)-2-yl]-L-2,3-di-O-docosyl-Sn-glycerol (200 mg) was reacted by thegeneral procedure according to Example 1 to obtain the title compound(237 mg, 83%) as white solid.

¹H-NMR (CD₃OD-D₂O, 1:1, 40° C.) δ: 2.93 (br.dd, 1H, H-3eq), 1.96 (s, 3H,NAc), 1.77 (br.t, 1H, H-3ax), 1.28 (m, 76H, 38CH₂), 0.87 (t, 6H, J=6.6Hz, 2CH₂CH₃).

Example 45

Methyl[2,2-bis(oleyl oxymethyl)propyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosid]onate

A 2,2-Bis(Oleyl oxymethyl)propanol

1,1,1-Tris(hydroxymethyl)ethane (1.0 g) and oleyl chloride (5.25 g) werereacted by the general procedure according to Example 15-A to obtain thetitle compound (2.91 g, 57%) as white solid.

¹H-NMR (CDCl₃) δ: 5.36 (m, 4H, 2CH═CH), 3.56 (d, 2H, J=5.9 Hz, CH₂OH),1.27 (m, 40H, 20CH₂), 0.88 (t, 6H, J=6.6 Hz, 2CH₂CH₃), 0.85 (s, 3H,CCH₃).

B Methyl[2,2-bis(oleyl oxymethyl)propyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosid]onate

2,2-Bis(oleyl oxymethyl)propanol (400 mg) and methyl5-acetamido-4,7,8,9-tetra-O-acetyl-2-hloro-2,3,5-trideoxy-D-glycero-β-D-galacto-2-nonulopyranosonate[Chem. Ber., 99, 611 (1966)] (328 mg) were reacted by the generalprocedure according to Example 15-C to obtain the title compound (564mg, 80%) as white solid.

¹H-NMR (CDCl₃) δ: 5.35 (m, 4H, 2CH═CH), 3.78 (s, 3H, COOCH₃), 2.58 (dd,1H, J=5.0, 12.8 Hz, H-3eq.), 2.13, 2.12, 2.04, 2.02, 1.88 (5s, 15H,5Ac), 1.27 (m, 40H, 20CH₂), 0.90 (s, 3H, CCH₃), 0.88 (t, 6H, J=6.9 Hz,2CH₂CH₃).

Example 46

2,2-Bis(oleyl oxymethyl)propyl5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosidonicacid:

Methyl[2,2-bis(oleyl oxymethyl)propyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosid]onate(250 mg) was reacted by the general procedure according to Example 16 toobtain the title compound (190 mg, 89%) as white solid.

¹H-NMR (CDCl₃-CD₃OD, 1:1) δ: 5.35 (m, 4H, 2CH═CH), 2.76 (dd, 1H, J=4.2,13.4 Hz, H-3eq), 2.05 (s, 3H, NAc), 1.70 (t, 1H, J=11.9 Hz, H-3ax), 1.30(m, 40H, 20CH₂), 0.92 (s, 3H, CCH₃), 0.89 (t, 6H, J=6.8 Hz, 2CH₂CH₃).

Example 47

Sodium[2,2-bis(oleyl oxymethyl)propyl5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosid)onate

2,2-Bis(oleyl oxymethyl)propyl5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosidonic acid(913 mg) was reacted by the general procedure according to Example 1 toobtain the title compound (63 mg, 47%) as white solid.

¹H-NMR (CD₃OD-D₂O, 1:1, 50° C.) δ: 5.37 (m, 4H, 2CH═CH), 2.95 (dd, 1H,J=5.3, 12.6 Hz, H-3eq), 1.97 (s, 3H, NAc), 1.74 (t, 1H, J=12.8 Hz,H-3ax), 1.28 (m, 40H, 20CH₂), 0.94 (s, 3H, CCH₃), 0.89 (t, 6H, J=6.8 Hz,2CH₂CH₃).

Example 48

Methyl[2,2-bis(docosyloxymethyl)butyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosid]onate

A 2,2-Bis(docosyloxymethyl)butanol

1,1,1-Tris(hydroxymethyl)propane (1.0 g) and 1-bromo docosane (7.3 g)were reacted by the general procedure according to Example 15-A toobtain the title compound (3.12 g, 56%) as white solid.

¹H-NMR (CDCl₃) δ: 3.59 (d, 2H, J=5.9 Hz, CH₂OH), 1.25 (m, 76H, 38CH₂),0.88 (t, 6H, J=6.7 Hz, 2CH₂CH₃), 0.84 (t, 3H, J=7.5 Hz, CCH₂CH₃).

B Methyl[2,2-bis(docosyloxymethyl)butyl5acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosid]onate

2,2-Bis(docosyloxymethyl)butanol (2.26 g) and methyl5-acetamido-4,7,8,9-tetra-O-acetyl-2-chloro-2,3,5-trideoxy-D-glycero-β-D-galacto-2-nonulopyranosonate[Chem. Ber., 99, 611 (1966)] (1.02 g) were reacted by the generalprocedure according to Example 15-C to obtain the title compound (539mg, 22%) as white solid.

¹H-NMR (CDCl₃) δ: 3.78 (s, 3H, COOCH₃), 2.58 (dd, 1H, J=4.8, 12.8 Hz,H-3eq.), 2.13, 2.12, 2.04, 2.02, 1.88 (5s, 15H, 5Ac), 1.95 (t, 1H,J=12.5 Hz, H-3ax.), 1.25 (m, 76H, 38CH₂), 0.88 (t, 6H, J=7.0 Hz,2CH₂CH₃), 0.84 (t, 3H, J=7.5 Hz, CCH₂CH₃).

Example 49

2,2-Bis(docosyloxymethyl)butyl5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosidonicacid:

Methyl[2,2-bis(docosyloxymethyl)butyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosid]onate(520 mg) was reacted by the general procedure according to Example 16 toobtain the title compound (393 mg, 90%) as white solid.

¹H-NMR (CDCl₃-CD₃OD, 1:1, 60° C.) δ: 2.72 (dd, 1H, J=5.1, 12.1 Hz,H-3eq), 2.02 (s, 3H, NAc), 1.80 (t, 1H, J=11.9 Hz, H-3ax), 1.28 (m, 76H,38CH₂), 0.89 (t, 6H, J=7.0 Hz, 2CH₂CH₃), 0.86 (t, 3H, J=7.7 Hz,CCH₂CH₃).

Example 50

Sodium[2,2-bis(docosyloxymethyl)butyl5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosid)onate

2,2-Bis(docosyloxymethyl)butyl5-acetamido-3,5-dideoxy-D-glycero-β-D-galacto-2-nonulopyranosidonic acid(388 mg) was reacted by the general procedure according to Example 1 toobtain the title compound (332 mg, 61%) as white solid.

¹H-NMR (CD₃OD-D₂O, 1:1, 60° C.) δ: 2.95 (br.dd, 1H, H-3eq), 1.96 (s, 3H,NAc), 1.78 (br.t, 1H, H-3ax), 1.27 (m, 76H, 38CH₂), 0.87 (t, 9H, J=7.0Hz, 3CH₂CH₃).

Example 51

3-O-[Methyl{5-N-(O-acetylglycolyl)-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl}onate]-1,2-di-O-docosyl-Sn-glycerol

[1,2-Di-O-docosyl-Sn-glycerol (400 mg) and methyl5-N-(O-acetylglycolyl)-4,7,8,9-tetra-O-acetyl-2-chloro-2,3,5-trideoxy-D-glycero-β-D-galacto-2-nonulopyranosonate[Carbohydr. Res., 174, 73 (1988)] (300 mg) were reacted by the generalprocedure according to Example 15-C to obtain the title compound (66 mg,10%) as white solid.

¹H-NMR (CDCl₃) δ: 4.60, 4.29 (2d, 2H, J=5.0 Hz, NHCOCH₂), 3.81 (s, 3H,OCH₃), 2.62 (dd, 1H, J=4.8, 12.8 Hz, H-3eq), 2.20, 2.15, 2.13, 2.04,2.01 (5s, 15H, 5OAc), 1.25 (m, 76H, 38CH₂), 0.88 (t, 6H, J=7.0 Hz,2CH₂CH₃).

Example 52

3-O-[(5-N-glycolyl-3,5-dideoxy-D-glycero-α-D-galacto-nonulopyranosonicacid)-2-yl]-1,2-di-O-docosyl-Sn-glycerol

3-O-[Methyl{5-N-(O-acetylglycolyl)-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl}onate]-1,2-di-O-docosyl-Sn-glycerol(60 mg) was reacted by the general procedure according to Example 16 toobtain the title compound (43 mg, 88%) as white solid.

¹H-NMR (CDCl₃-CD₃OD, 1:1, 60° C.) δ: 2.78 (dd, 1H, J=4.0, 12.0 Hz,H-3eq), 1.79 (t, 1H, J=12.0 Hz, H-3ax), 1.28 (m, 76H, 38CH₂), 0.89 (t,6H, J=7.0 Hz, 2CH₂CH₃).

Example 53

3-O-[Sodium{5-N-(O-sodiumoxysulfonylglycolyl)-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosyl}onate]-1,2-di-O-docosyl-Sn-glycerol

3-O-[{-N-glycolyl-3,5-dideoxy-D-glycero-α-D-galacto-nonulopyranosonicacid}-2-yl]-1,2-di-O-docosyl-Sn-glycerol (41 mg) was reacted by thegeneral procedure according to Example 1 to obtain the title compound(16 mg, 26%) as white solid.

¹H-NMR (CD₃OD-D₂O, 1:1, 60° C.) δ: 2.99 (br.dd, 1H, H-3eq), 1.92 (br.t,1H, H-3ax), 1.30 (m, 76H, 38CH₂), 0.90 (t, 6H, J=7.0 Hz, 2CH₂CH₃).

Example 54

3-S-[Methyl(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl)onate]-1,2-di-O-docosyl-Sn-thioglycerol

A 3-Bromo-3-deoxy-1,2-di-O-docosyl-Sn-glycerol

1,2-Di-O-docosyl-Sn-glycerol (200 mg, 0.28 mmol), N-bromosuccinimide (90mg, 0.51 mmol) and triphenylphosphine (170 mg, 0.65 mmol) were stirredin toluene (12 ml) at room temperature for 3 days. The reaction solutionwas condensed in vacuo, and the residue was purified by silica gelcolumn chromatography (with 20 g of gel, hexane/toluene, 3:2) to obtainthe title compound (168 mg, 77%) as white powder.

¹H-NMR (CDCl₃) δ: 1.25 (m, 76H, 38CH₂), 0.88 (t, 6H, J=7.0 Hz, 2CH₂CH₃).

B3-S-[Methyl(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl)onate]-1,2-di-O-docosyl-Sn-thioglycerol

Methyl5-acetamido-4,7,8,9-tetra-O-acetyl-2-S-acetyl-3,5-dideoxy-2-thio-D-glycero-α-D-galacto-2-nonulopyranosonate[J. Carbohydr. Chem., 5, 11 (1986)] (114 mg, 0.21 mmol) and sodiummethoxide (11 mg, 0.20 mmol) were stirred in anhydrous methanol (0.5 ml)at −10° C. for 1 h, and the reaction solution was evaporated to drynessin vacuo. To the residue were added a solution of3-bromo-3-deoxy-1,2-di-O-docosyl-Sn-glycerol (160 mg, 0.21 mmol) indehydrated dimethylformamide (1.0 ml) and toluene (1.0 ml), and themixture was stirred at room temperature for two days. The reactionsolution was diluted with chloroform, washed with a saturated sodiumchloride solution, and dried over anhydrous magnesium sulfate. After thesolvent was distilled off in vacuo, the residue were purified by silicagel column chromatography (27 g of gel, toluene-acetone, 5:1) to obtainthe title compound (85 mg, 34%) as white powder.

¹H-NMR (CDCl₃) δ: 3.79 (s, 3H, COOCH₃), 2.91 (dd, 1H, J=4.8, 13.0 Hz,H-3eq.), 2.72 (m, 2H, SCH₂), 2.15, 2.13, 2.04, 2.03, 1.88 (5s, 15H,5Ac), 2.00 (t, 1H, J=12.8 Hz, H-3ax.), 1.25 (m, 76H, 38CH₂), 0.88 (t,6H, J=7.1 Hz, 2CH₂CH₃).

Example 55

3-S-[(5-Acetamido-3,5-dideoxy-D-glycero-α-D-galacto-nonulopyranosonicacid)-2-yl]-1,2-di-O-docosyl-Sn-thioglycerol

3-S-[Methyl(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl)onate]-1,2-di-O-docosyl-Sn-thioglycerol(154 mg) was reacted by the general procedure according to Example 16 toobtain the title compound (115 mg, 86%) as white solid.

¹H-NMR (CDCl₃-CD₃OD, 1:1, 60° C.) δ: 2.94 (dd, 1H, J=4.6, 12.8 Hz,H-3eq), 2.84 (m, 2H, SCH₂), 2.02 (s, 3H, NAc), 1.84 (t, 1H, J=11.6 Hz,H-3ax), 1.29 (m, 76H, 38CH₂), 0.89 (t, 6H, J=6.9 Hz, 2CH₂CH₃).

Example 56

3-S-[Sodium{5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosyl}onate]-1,2-di-O-docosyl-Sn-thioglycerol

3-S-[(5-Acetamido-3,5-dideoxy-D-glycero-α-D-galacto-nonulopyranosonicacid)-2-yl]-1,2-di-O-docosyl-Sn-thioglycerol (422 mg) was reacted by thegeneral procedure according to Example 1 to obtain the title compound(29 mg, 45%) as white solid.

¹H-NMR (CD₃OD-D₂O, 1:1, 50° C.) δ: 3.06 (br.dd, 1H, H-3eq), 2.90 (m, 2H,SCH₂), 1.90 (s, 3H, NAc), 1.30 (m, 76H, 38CH₂), 0.88 (t, 6H, J=7.0 Hz,2CH₂CH₃).

Example 57

1-[N-(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-O-methyl-D-glycero-β-D-galacto-2-nonulopyranosonyl)amino]-2,2-bis(docosyloxymethyl)propane

A 2,2-Bis(docosyloxymethyl)propyl methanesulfonate

After a mixture of 2,2-bis(docosyloxymethyl)propanol (7.37 g; 10 mmol),methanesulfonyl chloride (1.3 ml; 16.8 mmol) and pyridine (150 ml) wasstirred at 70° C. for 1 h, the reaction mixture was poured into water.Precipitates thus obtained were collected by suction, washedsuccessively with water and acetone, and dried to obtain the titlecompound (6.66 g, 82%).

¹H-NMR (CDCl₃) δ: 4.13 (s, 2H, CH₂OMs), 2.98 (s, 3H, CH₃SO₂), 1.26 (m,76H, 38CH₂), 1.00 (s, 3H, CCH₃), 0.88 (t, 6H, J=7.0 Hz, 2CH₂CH₃).

B 1-Azido-2,2-bis(docosyloxymethyl)propane

After a mixture of 2,2-bis(docosyloxymethyl)propyl methanesulfonate(22.7 g; 27.8 mmol), sodium azide (0.27 g; 83.5 mmol), anddimethylformamide (150 ml) was stirred at 110° C. for 20 h, the reactionmixture was poured into water. Precipitates thus obtained were collectedby suction, washed successively with water and acetone, and dried toobtain the title compound (20.8 g; 98%).

¹H-NMR (CDCl₃) δ: 1.25 (m, 76H, 38CH₂), 0.89 (s, 3H, CCH₃), 0.88 (t, 6H,J=7.0 Hz, 2CH₂CH₃).

C 1-Amino-2,2-bis(docosyloxymethyl)propane

1-Azido-2,2-bis(docosyloxymethyl)propane (12.8 g; 16.8 mmol) andpalladium hydroxide on carbon (6.0 g) in tetrahydrofuran (100 ml) wasstirred under the hydrogen atmosphere at 50° C. for 3 h. The reactionsolution was filtered through celite, and the filtrate was evaporated todryness in vacuo. The residue was purified by silica gel columnchromatography (60 g of gel, chloroform/methanol, 10:1) to obtain thetitle compound (8 g, 65%) as white powder.

D Methyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-β-D-galacto-2-nonulopyranosidonicacid:

After a mixture of methyl5-acetamido-3,5-dideoxy-D-glycero-β-D-galacto-2-nonulopyranosidonic acid[Chem. Ber., 99, 611 (1966)] (150 mg; 0.464 mmol), acetic anhydride (2.3ml) and pyridine (2.3 ml) was stirred at room temperature for 7 h, thereaction solution was evaporated to dryness in vacuo. The residue wasdissolved in chloroform, and treated with Amberist-15 (H type) to obtainthe title compound (218 mg, 96%) as white powder.

¹H-NMR (CDCl₃) δ: 3.33 (s, 3H, OCH₃), 2.56 (dd, 1H, J=4.8, 13.2 Hz,H-3eq.), 2.16, 2.11, 2.06, 2.04, 1.91 (5s, 15H, 5Ac).

E1-[N-(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-O-methyl-D-glycero-β-D-galacto-2-nonulopyranosonyl)amino]-2,2-bis(docosyloxymethyl)propane

After a mixture of methyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-β-D-galacto-2-nonulopyranosidonicacid (260 mg; 0.53 mmol), 1-amino-2,2-bis(docosyloxymethyl) propane (390mg; 0.53 mmol), dicyclohexylcarbodiimide (165 mg; 0.80 mmol),1-hydroxybenztriazole (95 mg; 0.70 mmol) and chloroform (11 ml) wasstirred at room temperature for 17 h, the reaction solution was filteredthrough celite, an the solvent was distilled off in vacuo. The residuewas purified by silica gel column chromatography (with 60 g of gel,chloroform-acetone, 17:3) to obtain the title compound (620 mg, 97%) aswhite powder.

¹H-NMR (CDCl₃) δ: 3.18 (s, 3H, OCH₃), 2.51 (dd, 1H, J=4.8, 13.2 Hz,H-3eq), 2.1 4, 2.08, 2.03, 2.01, 1.90 (5s, 15H, 5Ac), 1.77 (t, 1H,J=11.7 Hz, H-3ax), 1.25 (m, 76H, 38CH₂), 0.91 (s, 3H, CCH₃), 0.88 (t,6H, J=6.6 Hz, 2CH₂CH₃).

Example 58

1-[N-(5-Acetamido-3,5-dideoxy-2-O-methyl-D-glycero-β-D-galacto-2-nonulopyranosonyl)amino]-2,2-bis(docosyloxymethyl)propane

After a mixture of1-[N-(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-O-methyl-D-glycero-β-D-galacto-2-nonulopyranosonyl)amino]-2,2-bis(docosyloxymethyl)propane (620 mg; 0.51 mmol), sodium methoxide (28 mg; 0.52 mmol),methanol (5 ml) and tetrahydrofuran (5 ml) was stirred at roomtemperature for 7 h, the reaction solution was neutralized withAmberlist-15 (H type), and evaporated to dryness in vacuo. The residuewas purified by silica gel column chromatography (50 g of gel,chloroform/methanol, 10:1) to obtain the title compound (480 mg, 90%) aswhite powder.

¹H-NMR (CDCl₃-CD₃OD, 1:1) δ: 3.24 (s, 3H, OCH₃), 2.40 (dd, 1H, J=4.9,13.0 Hz, H-3eq), 2.05 (s, 3H, NAc), 1.27 (m, 76H, 38CH₂), 0.91 (s, 3H,CCH₃), 0.89 (t, 6H, J=7.0 Hz, 2CH₂CH₃).

Example 59

1-[N-{5-Acetamido-3,5-dideoxy-2-O-methyl-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-β-D-galacto-2-nonulopyranosonyl}amino]-2,2-bis(docosyloxymethyl)propane

1-[N-(5-Acetamido-3,5-dideoxy-2-O-methyl-D-glycero-β-D-galacto-2-nonulopyranosonyl)amino]-2,2-bis(docosyloxymethyl)propane (831 mg) was reacted by the general procedure according toExample 1 to obtain the title compound (810 mg, 70%) as white solid.

¹H-NMR (CD₃OD-D₂O, 1:1, 40° C.) δ: 2.66 (br.dd, 1H, J=4.4, 12.5 Hz,H-3eq), 2.03 (s, 3H, NAc), 1.79 (br.t, 1H, H-3ax), 1.31 (m, 76H, 38CH₂),0.91 (s, 3H, CCH₃), 0.91 (t, 6H, J=6.8 Hz, 2CH₂CH₃).

Example 60

1-[N-(5-Aetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-O-methyl-D-glycero-β-D-galacto-2-nonulopyranosonyl)amino]-2,2-bis(eicosyloxymethyl)propane

A 2,2-Bis(eicosyl oxymethyl)propyl methanesulfonate

2,2-Bis(eicosyloxymethyl)propanol (1.19 g) was reacted by the generalprocedure according to Example 57-A to obtain the title compound (1.08g, 82%) as white solid.

¹H-NMR (CDCl₃) δ: 4.13 (s, 2H, CH₂OMs), 2.98 (s, 3H, CH₃SO₂), 1.25 (m,68H, 34CH₂), 1.00 (s, 3H, CCH₃), 0.88 (t, 6H, J=7.0 Hz, 2CH₂CH₃).

B 1-Azido-2,2-bis(eicosyloxymethyl)propane

2,2-Bis(eicosyloxymethyl)propyl methanesulfonate (1.05 g) was reacted bythe general procedure according to Example 57-B to obtain the titlecompound (0.94 g, 96%) as white solid.

¹H-NMR (CDCl₃) δ: 1.25 (m, 68H, 34CH₂), 0.94 (s, 3H, CCH₃), 0.88 (t, 6H,J=7.0 Hz, 2CH₂CH₃).

C 1-Amino-2,2-bis(eicosyloxymethyl) propane

1-Azido-2,2-bis(eicosyloxymethyl) propane (360 mg) was reacted by thegeneral procedure according to Example 57-C to obtain the title compound(206 mg, 59%) as white solid.

¹H-NMR (CDCl₃) δ: 2.74 (s, 2H, CH₂), 1.25 (m, 68H, 34CH₂), 0.90 (s, 3H,CCH₃), 0.88 (t, 6H, J=7.0 Hz, 2CH₂CH₃).

D1-[N-(5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-O-methyl-D-glycero-β-D-galacto-2-nonulopyranosonyl)amino]-2,2-bis(eicosyloxymethyl)propane

Methyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-β-D-galacto-2-nonulopyranosidonicacid (179 mg) and 1-amino-2,2-bis(eicosyloxymethyl)propane (206 mg) werereacted by the general procedure according to Example 57-E to obtain thetitle compound (327 mg, 94%) as white solid.

¹H-NMR (CDCl₃) δ: 3.18 (s, 3H, OCH₃), 2.51 (dd, 1H, J=5.0, 13.0 Hz,H-3eq), 2.14, 2.09, 2.04, 2.01, 1.91 (5s, 15H, 5Ac), 1.77 (dd, 1H,J=11.7, 12.8 Hz, H-3ax), 1.25 (m, 68H, 34CH₂), 0.91 (s, 3H, CCH₃), 0.88(t, 6H, J=7.0 Hz, 2CH₂CH₃).

Example 61

1-[N-(5-Acetamido-3,5-dideoxy-2-O-methyl-D-glycero-β-D-galacto-2-nonulopyranosonyl)amino]-2,2-bis(eicosyloxymethyl)propane

1-[N-(5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-O-methyl-D-glycero-β-D-galacto-2-nonulopyranosonyl)amino]-2,2-bis(eicosyloxymethyl)propane(216 mg) was reacted by the general procedure according to Example 58 toobtain the title compound (202 mg, 74%) as white solid.

¹H-NMR (CDCl₃-CD₃OD, 10:1) δ: 3.22 (s, 3H, OCH₃), 2.36 (dd, 1H, J=4.8,13.2 Hz, H-3eq), 2.04 (s, 3H, NAc), 1.26 (m, 68H, 34CH₂), 0.90 (s, 3H,CCH₃), 0.88 (t,6H, J=7.1 Hz, 2CH₂CH₃).

Example 62

1-[N-{5-Aetamido-3,5-dideoxy-2-O-methyl-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-β-D-galacto-2-nonulopyranosonyl}amino]-2,2-bis(eicosyloxymethyl)propane

1-[N-(5-Acetamido-3,5-dideoxy-2-O-methyl-D-glycero-β-D-galacto-2-nonulopyranosonyl)amino]-2,2-bis(eicosyloxymethyl)propane(422 mg) was reacted by the general procedure according to Example 1 toobtain the title compound (49 mg, 36%) as white solid.

¹H-NMR (CD₃OD-D₂O, 1:1, 40° C.) δ: 2.66 (br.dd, 1H, H-3eq), 2.02 (s, 3H,NAc), 1.78 (br.t, 1H, J=12.3 Hz, H-3ax), 1.30 (m, 68H, 34CH₂), 0.90 (s,3H, CCH₃), 0.90 (t, 6H, J=7.0 Hz, 2CH₂CH₃).

Example 63

1-[N-(5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-O-ethyl-D-glycero-β-D-galacto-2-nonulopyranosonyl)amino]-2,2-bis(docosyloxymethyl)propane

A Methyl(ethyl5-acetamido-3,5-dideoxy-D-glycero-β-D-galacto-2-nonulopyranosid)onate

A mixture of N-acetylneuraminic acid (1.00 g; 3.23 mmol), Dowex-50 (Hform) (15 g) and dehydrated ethanol (100 ml) was heated at reflux for 18h. The reaction mixture was packed into a column, and eluted with2N-HCl-methanol (100 ml). After the solvent was distilled off in vacuo.The residue thus obtained was stirred with acetic anhydride (20 ml) andpyridine (20 ml) at room temperature for 17 h, and then the reactionsolution was evaporated to dryness in vacuo. The residue was dissolvedin chloroform, washed successively with 0.1N HCl, water and saturatedNaCl solution, dried over anhydrous magnesium sulfate, and then thesolvent was distilled off in vacuo. After the residue thus obtained wasstirred with sodium methoxide (31 mg; 0.575 mmol) in methanol (23 ml) atroom temperature for 17 h, the reaction mixture was neutralized withDowex-50 (H form), and then the solvent was distilled off. The residuewas purified by silica gel column chromatography (40 g of gel,chloroform/methanol, 4:1) to obtain the title compound (142 mg, 12%) aswhite powder.

¹H-NMR (CD₃OD) δ: 3.78 (s, 3H, OCH₃), 2.36 (dd, 1H, J=4.9, 13.0 Hz,H-3eq.), 2.00 (s, 3H, NAc), 1.62 (dd, 1H, J=11.4, 12.8 Hz, H-3ax.), 1.16(t, 3H, J=7.0 Hz, CH₂CH₃).

B Methyl(ethyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-β-D-galacto-2-nonulopyranosid)onate

Methyl(ethyl5-acetamido-3,5-dideoxy-D-glycero-β-D-galacto-2-nonulopyranosid)onate(142 mg) was reacted by the general procedure according to Example 57-Dto obtain the title compound (209 mg, 99%) as white solid.

¹H-NMR (CDCl₃) δ: 3.80 (s, 3H, COOCH₃), 2.45 (dd, 1H, J=5.1, 12.8 Hz,H-3eq.), 2.15, 2.08, 2.03, 2.02, 1.89 (5s, 15H, 5Ac), 1.87 (dd, 1H,J=11.4, 12.8 Hz, H-3ax.), 1.22 (t, 3H, J=7.1 Hz, CH₂CH₃).

C Ethyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-β-D-galacto-2-nonulopyranosidonicacid:

A mixture of methyl (ethyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-β-D-galacto-2-nonulopyranosid)onate(203 mg, 0.39 mmol), anhydrous lithium iodide (523 mg, 3.9 mmol) andpyridine (8.0 ml) was stirred at 90° C. for 14 h. The reaction solutionwas subjected to gel filtration (LH-20, 150 ml, methanol), and thenpurified by silica gel column chromatography (20 g of gel,chloroform/methanol, 7:3) to obtain the title compound (92 mg, 47%) aswhite solid.

¹H-NMR (CDCl₃) δ: 3.52 (m, 2H, CH₂CH₃), 2.56 (dd, 1H, J=4.5, 12.2 Hz,H-3eq.), 2.15, 2.11, 2.06, 2.04, 1.91 (5s, 15H, 5Ac), 1.92 (t, 1H,J=11.8 Hz, H-3ax.), 1.24 (t, 3H, J=7.0 Hz, CH₂CH₃).

D1-[N-(5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-O-ethyl-D-glycero-β-D-galacto-2-nonulopyranosonyl)amino]-2,2-bis(docosyloxymethyl)propane

Ethyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-β-D-galacto-2-nonulopyranosodonicacid (92 mg) and 1-amino-2,2-bis(docosyloxymethyl)propane (268 mg) werereacted by the general procedure according to Example 57-E to obtain thetitle compound (191 mg, 86%) as white solid.

¹H-NMR (CDCl₃) δ: 2.52 (dd, 1H, J=4.8, 13.2 Hz, H-3eq), 2.14, 2.08,2.03, 2.01, 1.91 (5s, 15H, 5Ac), 1.76 (t, 1H, J=12.4 Hz, H-3ax), 1.25(m, 76H, 38CH₂), 1.19 (t, 3H, J=7.0 Hz, OCH₂CH₃), 0.90 (s, 3H, CCH₃),0.88 (t, 6H, J=7.0 Hz, 2CH₂CH₃).

Example 64

1-[N-(5-Acetamido-3,5-dideoxy-2-O-ethyl-D-glycero-β-D-galacto-2-nonulopyranosonyl)amino]-2,2-bis(docosyloxymethyl)propane

1-[N-(5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-O-ethyl-D-glycero-β-D-galacto-2-nonulopyranosonyl)amino]-2,2-bis(docosyloxymethyl)propane(185 mg) was reacted by the general procedure according to Example 58 toobtain the title compound (114 mg, 71%) as white solid.

¹H-NMR (CDCl₃-CD₃OD, 10:1) δ: 2.38 (dd, 1H, J=4.8, 13.2 Hz, H-3eq), 2.04(s, 3H, NAc), 1.26 (m, 76H, 38CH₂), 1.17 (t, 3H, J=7.0 Hz, OCH₂CH₃),0.89 (s, 3H, CCH₃), 0.88 (t, 6H, J=7.3 Hz, 2CH₂CH₃).

Example 65

1-[N-{5-Acetamido-3,5-dideoxy-2-O-ethyl-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-β-D-galacto-2-nonulopyranosonyl}amino]-2,2-bis(docosyloxymethyl)propane

1-[N-(5-Acetamido-3,5-dideoxy-2-O-ethyl-D-glycero-β-D-galacto-2-nonulopyranosonyl)amino]-2,2-bis(docosyloxymethyl)propane (422 mg) was reacted by the general procedure according toExample 1 to obtain the title compound (140 mg, 93%) as white solid.

¹H-NMR (CD₃OD-D₂O, 1:1, 40° C.) δ: 2.67 (br.dd, 1H, H-3eq), 2.02 (s, 3H,NAc), 1.77 (br.t, 1H, H-3ax), 1.30 (m, 76H, 38CH₂), 1.25 (t, 3H, J=7.0Hz, OCH₂CH₃), 0.91 (t, 6H, J=6.8 Hz, 2CH₂CH₃), 0.90 (s, 3H, CCH₃).

Example 66

1-[N-(5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-O-methyl-D-glycero-α-D-galacto-2-nonulopyranosonyl)amino]-2,2-bis(docosyloxymethyl)propane

A Methyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosidonicacid:

Methyl5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosidonic acid[Chem. Ber., 99, 611(1966)] (150 mg) was reacted by the generalprocedure according to Example 57-D to obtain the title compound (220mg, 96%) as white solid.

¹H-NMR (CDCl₃-CD₃OD, 1:1) δ: 3.20 (s, 3H, OCH₃), 2.42 (dd, 1H, J=4.4,12.5 Hz, H-3eq.), 1.96, 1.95, 1.87, 1.83, 1.70 (5s, 15H, 5Ac), 1.56 (t,1H, J=12.5 Hz, H3ax.).

B1-[N-(5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-O-methyl-D-glycero-α-D-galacto-2-nonulopyranosonyl)amino]-2,2-bis(docosyloxymethyl)propane

Methyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-ct-D-galacto-2-nonulopyranosidonicacid (191 mg) and 1-amino-2,2-bis(docosyloxymethyl) propane (396 mg)were reacted by the general procedure according to Example 57-E toobtain the title compound (345 mg, 73%) as white solid.

¹H-NMR (CDCl₃) δ: 3.36 (s, 3H, OCH₃), 2.13, 2.08, 2.03, 2.01, 1.89 (5s,15H, 5Ac), 1.98 (t, 1H, J=11.7 Hz, H3ax.) 1.25 (m, 76H, 38CH₂), 0.90 (s,3H, CCH₃), 0.88 (t, 6H, J=7.0 Hz, 2CH₂CH₃).

Example 67

1-[N-(5-Acetamido-3,5-dideoxy-2-O-methyl-D-glycero-α-D-galacto-2-nonulopyranosonyl)amino]-2,2-bis(docosyloxymethyl)propane

1-[N-(5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-O-methyl-D-glycero-α-D-galacto-2-nonulopyranosonyl)amino]-2,2-bis(docosyloxymethyl)propane(340 mg) was reacted by the general procedure according to Example 58 toobtain the title compound (216 mg, 74%) as white solid.

¹H-NMR (CDCl₃-CD₃OD, 10:1) δ: 3.36 (s, 3H, OCH₃), 2.55 (dd, 1H, J=4.8,13.2 Hz, H-3eq), 2.03 (s, 3H, NAc), 1.82 (dd, 1H, J=11.2, 13.0 Hz,H3ax.), 1.26 (m, 76H, 38CH₂), 0.91 (s, 3H, CCH₃), 0.88 (t, 6H, J=7.0 Hz,2CH₂CH₃).

Example 68

1-[N-{5-Acetamido-3,5-dideoxy-2-O-methyl-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosonyl}amino]-2,2-bis(docosyloxymethyl)propane

1-[N-(5-Acetamido-3,5-dideoxy-2-O-methyl-D-glycero-α-D-galacto-2-nonulopyranosonyl)amino]-2,2-bis(docosyloxymethyl)propane(117 mg) was reacted by the general procedure according to Example 1 toobtain the title compound (98 mg, 60%) as white solid.

¹H-MR (CD₃OD-D₂O, 1:1, 40° C.) δ: 2.55 (br.dd, 1H, J=5.1, 13.6 Hz,H-3eq), 1.99 (s, 3H, NAc), 1.30 (m, 76H, 38CH₂), 0.90 (br.t, 9H, J=6.6Hz, CCH₃ and 2CH₂CH₃).

Assessment of Growth Inhibitory Effect on HIV-1 and Cytotoxicity

Samples of various concentrations (25 μl) are distributed into a 24-wellmicroplate, immediately added with human T cells (H9 cell line,1.2×10⁵/ml) (1 ml), covered, and incubated at 37° C. for 1 h. Then, AIDSvirus (HIV-1, IIIb, 100 TCID 50) (25 μl) is added to each well andthoroughly mixed, covered again, and incubated under the CO₂ atmosphereat 37° C. for 3 days. On the fourth day, aliquots (200 μl) of theculture medium from each well are transferred into a fresh microplate.The growth medium (500 μl) is added to each well, and further incubatedfor 2 days.

On the 6th day, aliquots (90 μl) of the culture supernatant arewithdrawn, and assayed the amount of p24, a constituent of HIV-1, byELISA method using the anti-p24 antibody to assess the growth of saidvirus. Also, using the remaining cell culture suspension (100 μl),viable cells are counted by the MTT method. Experimental values areexpressed as the ratios (%) to the negative control value (measuredusing the solvent to dissolve test compounds as the sample) to representthe anti-HIV activity and cytotoxicity. Then, for the unifiedrepresentation of the anti-HIV activity and cytotoxicity, theconcentrations of test compounds at their 50% values are obtained fromthe activity-concentration curves and the cytotoxicity-concentration,and represented as IC50 and CC50, respectively.

Results of the Anti-HIV Activity Assay

In the following table are shown the compounds and their structuralcharacteristics having IC50 (μM) values less than 100 μM. Carbon numberof the functional group (in parentheses) represents that of alkyl groupof lipid side-chain beyond the ether linkage, and, particularly, in thecase where lipid is alkylglycerol, that of alkyl group of said alkylglycerol.

TABLE 1 Example No. IC 50 (μM) Functional group (carbon number) 5 46.0Two alkyl chains (10) 6 50.0 Two alkyl chains (10), β-isomer of 5 7 16.0Two alkyl chains (14) 8 15.0 Two alkyl chains (14) β-isomer of 7 9 3.8Two alkyl chains (18) 10 30.0 One alkyl chain (18), with a sulfate groupas a side chain 11 13.0 One alkyl chain (18), β-isomer of 10 12 0.8 Twoalkyl chains (22) 13 1.6 Two alkyl chains (22), β-isomer of 12 14 15.0Two alkyl chains (14), thioglycoside 17 2.5 Two alkyl chains (20) 20 1.2Two alkyl chains (24) 24 34.0 Cholesterol 25 100.0 One alkyl chain (18),unsaturated 26 100.0 One alkyl chain (18) 29 1.6 Two alkyl chains (22),branched 32 2.5 Two alkyl chains (20), branched 35 90.0 Aromatic 38 2.5Two alkyl chains (22), glycero-ester 41 4.0 Two alkyl chains (22), KDN50 1.1 Two alkyl chains (22), with an ethyl group at the branching point59 1.4 Two alkyl chains (22), amide linkage

These results indicate that, as the lipid bound to nonulonic acid(sialic acid and KDN) via glycosidic linkage are preferred alkyl,carbonyloxy or oxycarbonyl groups. Furthermore, no significantdifference observed in the activity of Example 7 (O-glycoside) ascompared with that of Example 14 (S-glycoside) indicates that thelinkage between carbohydrate and lipid moieties can be eitherO-glycosidic or S-glycosidic. Also, no difference observed in theactivity between Example 29 and Example 59, wherein they differ only inthat the former has the O-glycosidic linkage and the latter the amidelinkage, leads to the conclusion that the linkage between carbohydrateand lipid can be of any form.

So far as sulfuric acid derivatives of sialoglycerolipids are concerned,as evidently seen comparing Example 9 (two alkyl chains, each having 18carbon atoms, IC50: 3.8) and Example 11 (one alkyl chain with 18 carbonatoms, IC50: 13.0), both derivatives, one with single chain, and theother with two chains, have the enough activity for the practical use,but the latter one is generally more active. Therefore, derivatives withtwo alkyl chains can be more preferable than those with one alkyl chain.

Also, as evidently seen comparing Example 38 (two alkyl chains, eachhaving 22 carbon atoms, glyceroester, IC50: 2.5) and Example 12 (twoalkyl chains, each having 22 carbon atoms, IC50: 0.8), these compoundshave high activity regardless of whether their glycero-lipid moietiesare either alkylglycerol or acylglycerol. Therefore, the biologicalactivity is not so much affected by the linkage form between theglycerol skeleton and the long alkyl chain group, but rather mainlydependent on the length of long alkyl chain group.

So far as compounds wherein glycerol having two alkyl chains is linkedto sugar via glycosidic linkage are concerned, as shown in Table 1,minimum IC50 values are associated mostly with those with carbon atomsof 18 (Example 9), 20 (Example 17), 22 (Example 12) and 24 (Example 20).Therefore, in the case where the sulfate derivatives ofsialoglycerolipids related to this invention are either alkyl glycerolor acyl glycerol, number of carbon atoms of alkyl groups linked to theglycerol moiety is thought to be preferably 18˜24. Since the biologicalactivity depends mainly on the length of long-chain alkyl group linkedto the glycerol skeleton, said long chain alkyl group can be saturatedor unsaturated, also branched. In the case of alkyl glycerol with 24carbon atoms as in Example 20, the “number of skeleton-forming atoms”per one side chain amounts to 25. This is because the “number ofskeleton-forming atoms” includes the oxygen atom related to the etherlinkage, in addition to carbon atoms of alkyl group. Furthermore, in thecase of pseudo-glycerol as in Example 29 and Example 32, since thecarbon atom of methylene group between the carbon atom at the β positionof the main chain of glycerol moiety and the oxygen atom of the etherlinkage is also included in the “number of skeleton-forming atoms”, thatnumber becomes 24 in the case of Example 29, and 22 in the case ofExample 32.

Also, the glycerol moiety can have a methyl branch, and a methylenegroup between the main chain of the glycerol moiety and ether linkage orester linkage (Example 29 and Example 32). Therefore, the glycerolmoiety can have a short alkyl chain such as methyl, ethyl, propyl, butylgroups, etc. at the β carbon atom of its main chain (in this case,however, these groups cannot be called side chain or branched chainbecause of insufficient length of alkyl groups.), and one to threecarbon atoms can be present between the main chain of glycerol moietyand the ether or ester linkage.

In comparison of Example 29 with Example 12, two chains extending fromthe β carbon atom of the main chain of glycerol moiety are of the samelength in Example 29, while they are of the different length in Example12. That is, in Example 29, number of “skeleton-forming atom” per onechain of the two chains extending from the β carbon atom of the mainchain of glycerol moiety is the same 24, but different in Example 12, as23 and 24. Therefore, it can be concluded that, in the case of twochains, number of “skeleton-forming atoms” per one chain, furthermore,number of alkyl chain-forming carbon atoms can be different. Inaddition, as to Example 29, it is advantageous that the two chains arecompletely the same having the same component atoms and configurationbecause for the easy preparation.

In the table below are shown CC50s.

TABLE 2 Example No. CC 50 (μM) Example No. CC 50 (μM) 5 >200 20 >206 >200 24 >200 7 >200 25 >200 8 >200 26 >200 9 >200 29 >20 10 >20032 >200 11 200 35 >20 12 >200 38 >200 13 >200 41 >200 14 >200 50 >20017 >20 59 >200

These results indicate that compounds related to the present inventionare generally low in the cytotoxicity.

Assay of Antiviral Activity against other Viruses than HIV

Antiviral activity against human Parainfluenza virus, Respiratorysyncytial virus (RSV) and Herpes simplex II virus (HSVII) was determinedby the plaque reduction assay, while that against Felineimmunodeficiency virus (FIV) and Feline leukemia virus (FeLV) by thereverse transcriptase assay and ELISA, respectively.

1) Plaque Reduction Assay

a) Preparation Viruse, Host Cell and Virus Stock

Each virus was obtained from ATCC. As host cells, VERO cells were usedfor Parainfluenza virus, and HEP2 cells for other viruses.

To fibroblasts growing in a T150 flask were added virus-infected cells,and the cells were incubated until they reached 60-80% infection. Cellswere trypsinized, recovered and used as the virus stock. As the cellculture medium was used E-MEM containing 2-5% FBS, 100 U/ml Penicillin,2.5 μg/ml Aniphotericin and 10 μg/ml Gentamycin.

b) Preparation of Sample

Compound related to Example 29 was appropriately diluted to theconcentrations of 500, 100, 50, 10 and 5 μg/ml with the culture mediumor methyl cellulose. As the reference drug for HSV II was usedAcyclovir.

c) Assay Procedure

Anti-viral Activity

After cells were cultured in monolayer in 24-well microplates, thesupernatant was discarded, replaced with a diluted sample (compound)solution (0.5 ml), and the mixture was incubated at 36-38° C. under the5-7% CO₂ atmosphere for 1 h. At the same time, to the cell control welland virus control wells was added the equal amount of medium.

After the supernatant was discarded, the medium (0.5 ml) and the virusstock (0.2 ml) were added to the sample well and virus well,respectively. After incubating again for 1 h, the sample solution (1 ml)diluted with methylcellulose containing FBS was overlayered. To thecontrol well was added only the same amount of the vehicle.

Cells were cultured at 36-38° C. under the 5-7% CO₂ atmosphere, andexamined for the appearance of plaques under microscope. Monolayer ofcells was fixed with 10% formalin, washed with water, stained with 0.8%crystal violet, and dried. Number of plaques counted in the sample wellwas compared with that in the virus control well, and the decrease inthe number of plaques was used to express the antiviral activity.

Cytotoxicity Assay

Of the anti-viral activity assay procedures mentioned above, the virusinfection step was omitted from sample wells, and cells were cultured inthe presence of samples at various concentrations, and viable cellnumber was compared with that in the cell control well. Surviving cellnumber was counted by the tetrazolium method for the microplate assay.

2) Reverse Transcriptase Assay

a) Preparation of Host Cell and Virus

Each virus was obtained from ATCC. FIV was cultured with CRFK cells inEagle's balanced salt medium containing 10% FBS, and the supernatant wasused as the virus stock. As FeLV was used ATCC VR-717.

b) Preparation of Sample

Compound related to Example 29 was diluted with the medium to similarconcentrations described above.

c) Assay Procedure

For each single strain of virus, the assay was performed in thefollowing combinations of conditions.

Medium control well Culture medium Cell control well Culture medium +cell Cytotoxicity well Culture medium + cell + sample Virus control wellCulture medium + cell + virus test well Culture medium + cell + virus +sample Color control well Culture medium + sample

After CRFL cells were cultured in monolayer in 96-well microplates, thesupernatant was discarded. Then, to the medium control well, the cellcontrol well and the virus control well was added the medium (0.2 ml),and to the cytotoxicity well, the test well and the color control wellwas added the diluted sample solution at 0.2, 0.1, and 0.2 ml,respectively, and incubated under the 5-7% CO₂ atmosphere at 36-38° C.for 1 h. Then, supernatants of the virus control well and the test wellwere discarded, and the virus stock (0.1 ml) was added, and incubatedfurther for 1 h. The, after supernatants were removed, and, to the viruscontrol well was added the medium (0.2 ml), and to the test well thesame volume of the diluted sample, then the incubation was furthercontinued.

Reverse Transcriptase Assay by ELISA

Using supernatants as the sample from the cell control well, the viruscontrol well and the test well, the reverse transcriptase of FIV or FeLVantigen contained therein were quantitated using assay kits fromAmersham LIFESCIENCE and Synbiotics, respectively. Anti-viral activitywas expressed as that ratio (%) of the value of the test well to that ofthe virus control well after both values were subtracted with the valueof the cell control well, respectively.

Cytotoxicity Assay

Viable cell numbers in the cytotoxicity well and the cell control wellwere counted by the tetrazolium method for microplate assay similarly asdescribed above, and the cytotoxicity was expressed as the ratio (%) ofthe cell number of the former to that of the latter.

Anti-viral Activity other than HIV and Cytotoxicity

Results of anti-viral activity assay are shown in the table below.

TABLE 3 Anti-viral activity of compound related to Example 29 IC50 (μM)Virus Example 29 Acyclovir Herpes simplex II virus 45.2 21.6 Respiratorysyncytial virus 5.1 — Parainfluenza virus 37.6 — Feline immunodeficiencyvirus 4.5 — Feline leukemia virus 58.3 —

These results indicate that the compound related to this invention hasnot only the anti-HIV activity but also the anti-viral activity againstother viruses.

Furthermore, when CC50s were calculated for each virus by the similarmethod as described above, they all exceeded the value 500 μM. Compoundsrelated to this invention have the anti-viral activity against otherviruses and low cytotoxicity, indicating that they are not onlyeffective in treating various diseases but also highly safe in theliving body and effective as therapeutics.

Anti-coagulation Activity

To the human plasma (400 μl) were added compound diluted withphysiological saline (500 μl) followed by 2% calcium chloride solution(100 μl), and the mixture was incubated at 37° C. for 30 min todetermine the minimum concentration (μM) of said compound to exhibit theanti-coagulant activity. Plasma coagulation was judged with the nakedeye.

Toxicity Study

Compounds were intravenously administered to mice, and LD50s (mg/kg)were determined.

Results of Anticoagulant Activity Assay and Toxicity Study

TABLE 4 Minimum concentration exhibiting anticoagulant action (μM) LD50(mg/kg) Dextran sulfate 0.5 — Example 12 900 2000 Example 13 1200 2000Example 29 900 2000

These results indicate that compounds related to this invention aregenerally low not only in the cytotoxicity but also in the anticoagulantaction, especially with compounds related to Examples 12, 13 and 29,furthermore they are low in the toxicity compounds related to Examples12, 13 and 29, furthermore they are low in the toxicity to the livingbody. Therefore, it is obvious that pharmaceutics containing compoundsrelated to this invention at the effective doses are preferable asdrugs.

Effective dose of compounds related to this invention or the saltsthereof can be determined by the general method well known to thoseskilled in the art including the method of establishing thedose-reaction curve in appropriate animal model or non-human primate andextrapolating its data to humans, or that of determining the dose in theclinical test.

Preferable doses of drugs, anti-viral agents and anti-HIV agents relatedto this invention are varied by various factors such as the severity ofdisease, body weight and age of individuals, half life of drug in theblood circulation, etc., and they can be easily determined by thoseskilled in the art.

Medicine related to this invention can be administered by various wayssuch as intravenous injection, oral administration, inhalation, etc.Pharmaceutical carriers, diluents and excipients can be easily selectedby those skilled in the art according to the clinical use of drug, and,if necessary, supplements such as disintegrator, binder (includingliposome), surfactant, emulsifier, buffer, solubilizing agent orpreservative are added to make liquid preparation, emulsion orsuspension.

As described above, novel compounds related to this invention havesignificant effectiveness such as not only the high antiviral activitybut also the low cytotoxicity. Therefore, novel compounds related to thepresent invention are optimal as the antiviral agent.

Also, since novel compounds related to the present invention are low notonly in the anticoagulant action but also in the toxicity to the livingbody, pharmaceutical preparations containing them in effective doses arepreferable as medicine. In view of the bleeding tendency observedparticularly with HIV patients, it is obvious that these novel compoundsare extremely useful as the anti-HIV medicine.

What is claimed is:
 1. A compound according to formula (I) or a saltthereof:

wherein Ac is acetyl; X is selected from the group consisting of oxygenand sulfur; Y is selected from the group consisting of H and an alkylgroup having from 1 to 4 carbon atoms; and each R is independentlyselected from the group consisting of branched and linear alkyl chainshaving from 10 to 24 carbon atoms.
 2. The compound according to claim 1,wherein Y is selected from the group consisting of H, CH₃ and CH₂CH₃. 3.The compound according to claim 2, wherein Y is selected from the groupconsisting of H and CH₃.
 4. A pharmaceutical composition comprising thecompound or salt of claim 3 and a pharmaceutically acceptable carrier.5. A pharmaceutical composition comprising the compound or salt of claim2 and a pharmaceutically acceptable carrier.
 6. The compound accordingto claim 1, wherein X is O.
 7. A pharmaceutical composition comprisingthe compound or salt of claim 6 and a pharmaceutically acceptablecarrier.
 8. The compound according to claim 1, wherein each R is thesame.
 9. The compound according to claim 8, wherein each R is a linearalkyl chain.
 10. The compound according to claim 9, wherein each R hasfrom 22 to 24 carbon atoms.
 11. A pharmaceutical composition comprisingthe compound or salt of claim 10 and a pharmaceutically acceptablecarrier.
 12. A pharmaceutical composition comprising the compound orsalt of claim 9 and a pharmaceutically acceptable carrier.
 13. Apharmaceutical composition comprising the compound or salt of claim 8and a pharmaceutically acceptable carrier.
 14. The compound according toclaim 1, selected from the group consisting of:3-O-(sodium(5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosyl)onate)-1,2-di-O-docosyl-Sn-glycerol,3-O-(sodium(5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-β-D-galacto-2-nonulopyranosyl)onate)-1,2-di-O-docosyl-Sn-glycerol,sodium(2,2-bis(docosyloxymethyl)butyl5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosid)onate, and1-(N-(5-acetamido-3,5-dideoxy-2-O-methyl-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-β-D-galacto-2-nonulopyranosonyl)amino-2,2-bis(docosyloxymethyl) propane.
 15. A pharmaceuticalcomposition comprising the compound or salt of claim 14 and apharmaceutically acceptable carrier.
 16. A pharmaceutical compositioncomprising the compound or salt of claim 1 and a pharmaceuticallyacceptable carrier.
 17. A method of treating a virus-mediated disease ina mammal comprising administering to said mammal a compound according toclaim 1, or a salt thereof.
 18. The method according to claim 17,wherein said compound is administered to said mammal by a route selectedfrom the group consisting of an intravenous route, an intramuscularroute, an intraperitoneal route, an intra-arterial route, a subcutaneousroute, a percutaneous route, a sublingual route, an inhalation route, arectal route, and an oral route.
 19. The method according to claim 17,wherein said virus is selected from the group consisting of a humanimmonodeficiency virus, a feline immunodeficiency virus, a felineleukemia virus, a herpes simplex II virus, a parainfluenza virus, arespiratory syncytial virus, and combinations thereof.
 20. The methodaccording to claim 19, wherein said virus is a respiratory syncytialvirus.
 21. The method according to claim 19, wherein said virus is ahuman immonodeficiency virus.
 22. A compound or salt thereof selectedfrom the group consisting of:3-O-(sodium(5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosyl)onate)-1,2-di-Odecyl-Sn-glycerol,3-O-(sodium(5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-β-D-galacto-2-nonulopyranosyl)onate)-1,2-di-O-decyl-Sn-glycerol,3-O-(sodium(5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosyl)onate)-1,2-di-O-tetradecyl-Sn-glycerol,3-O-(sodium(5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-β-D-galacto-2-nonulopyranosyl)onate)-1,2-di-O-tetradecyl-Sn-glycerol,3-O-(sodium(5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosyl)onate)-1,2-di-O-octadecyl-Sn-glycerol,3-O-(sodium(5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosyl)onate)-2-O-(sodiumoxysulfonyl)-1-O-octadecyl-Sn-glycerol,3-O-(sodium(5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-β-D-galacto-2-nonulopyranosyl)onate)-2-O-(sodiumoxysulfonyl)-1-O-octadecyl-Sn-glycerol,3-O-(sodium(5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosyl)onate)-1,2-di-O-docosyl-Sn-glycerol,3-O-(sodium(5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-f3-D-galacto-2-nonulopyranosyl)onate)-1,2-di-O-docosyl-Sn-glycerol,3-S-(sodium(5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosyl)onate)-1,2-di-O-tetradecyl-Sn-thioglycerol,3-O-(sodium(5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosyl)onate)-1,2-di-O-eicosyl-Sn-glycerol,3-O-(sodium(5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosyl)onate)-1,2-di-O-tetracosyl-Sn-glycerol,3β-(sodium(5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosid)onate, sodium(oleyl5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosid)onate,sodium(octadecyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-galacto-2-nonulopyranosid)onate,sodium(2,2-bis(eicosyl oxymethyl)propyl5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosid)onate,sodium(3,5-didocosyloxyphenyl-5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-β-D-galacto-2-nonulopyranosid)onate,3-O-(sodium(5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-galacto-2-nonulopyranosyl)onate)-1,2-di-O-docosanoyl-Sn-glycerol,3-O-(sodium(3-deoxy-4,5,7,8,9-penta-O-(sodiumoxysulfonyl)-D-glycero-a-D-galacto-2-nonulopyranosyl)onate)-1,2-di-O-docosyl-Sn-glycerol,sodium(2,2-bis(docosyloxymethyl)butyl5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-α-D-galacto-2-nonulopyranosid)onate,1-(N-(5-acetamido-3,5-dideoxy-2-O-methyl-4,7,8,9-tetra-O-(sodiumoxysulfonyl)-D-glycero-β-D-galacto-2-nonulopyranosonyl)amino)-2,2-bis(docosyloxymethyl)propane, and salts thereof.
 23. A pharmaceutical composition comprisingthe compound or salt of claim 22 and a pharmaceutically acceptablecarrier.
 24. A method of treating a virus-mediated disease in a mammalcomprising administering to said mammal a compound according to claim22, or a salt thereof.
 25. The method according to claim 24, whereinsaid compound is administered to said mammal by a route selected fromthe group consisting of an intravenous route, an intramuscular route, anintraperitoneal route, an intra-arterial route, a subcutaneous route, apercutaneous route, a sublingual route, an inhalation route, a rectalroute, and an oral route.
 26. The method according to claim 24, whereinsaid virus is selected from the group consisting of a humanimmonodeficiency virus, a feline immunodeficiency virus, a felineleukemia virus, a herpes simplex II virus, a parainfluenza virus, arespiratory syncytial virus, and combinations thereof.
 27. The methodaccording to claim 26, wherein said virus is a respiratory syncytialvirus.
 28. The method according to claim 26, wherein said virus is ahuman immonodeficiency virus.